Transgenic mammals and methods of use thereof

ABSTRACT

The present invention relates to transgenic mammals that express canine-based immunoglobulins, including transgenic rodents that express canine-based immunoglobulins for the development of canine therapeutic antibodies.

FIELD OF THE INVENTION

This invention relates to production of immunoglobulin molecules,including methods for generating transgenic mammals capable of producingcanine antigen-specific antibody-secreting cells for the generation ofmonoclonal antibodies.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods are describedfor background and introductory purposes. Nothing contained herein is tobe construed as an “admission” of prior art. Applicant expresslyreserves the right to demonstrate, where appropriate, that the articlesand methods referenced herein do not constitute prior art under theapplicable statutory provisions.

Antibodies have emerged as important biological pharmaceuticals becausethey (i) exhibit exquisite binding properties that can target antigensof diverse molecular forms, (ii) are physiological molecules withdesirable pharmacokinetics that make them well tolerated in treatedhumans and animals, and (iii) are associated with powerful immunologicalproperties that naturally ward off infectious agents. Furthermore,established technologies exist for the rapid isolation of antibodiesfrom laboratory animals, which can readily mount a specific antibodyresponse against virtually any foreign substance not present natively inthe body.

In their most elemental form, antibodies are composed of two identicalheavy (H) chains that are each paired with an identical light (L) chain.The N-termini of both H and L chains consist of a variable domain (V_(H)and V_(L), respectively) that together provide the paired H-L chainswith a unique antigen-binding specificity. The exons that encode theantibody V_(H) and V_(L) domains do not exist in the germ-line DNA.Instead, each V_(H) exon is generated by the recombination of randomlyselected V, D, and J gene segments present in the H chain locus (Igh;see schematic of the mouse Igh locus in FIG. 1); likewise, individualV_(L) exons are produced by the chromosomal rearrangements of randomlyselected V and J gene segments in a light chain locus. The canine genomecontains two alleles that can express the H chain (one allele from eachparent), two alleles that can express the kappa (κ) L chain, and twoalleles that can express the lambda (λ) L chain. There are multiple V,D, and J gene segments at the H chain locus as well as multiple V and Jgene segments at both L chain loci. Downstream of the J genes at eachimmunoglobulin (Ig) locus exists one or more exons that encode theconstant region of the antibody. In the heavy chain locus, exons for theexpression of different antibody classes (isotypes) also exist. Incanine animals, the encoded isotypes are IgM, IgD, IgG1, IgG2, IgG3,IgG4, IgE, and IgA. Polymorphic variants (referred to as allotypes) alsoexist among canine strains for IgG2, IgE and IgA and are useful asallelic markers.

During B cell development, gene rearrangements occur first on one of thetwo homologous chromosomes that contain the H chain variable genesegments. The resultant V_(H) exon is then spliced at the RNA level tothe exons that encode the constant region of the H chain (C_(H)).Subsequently, the VJ rearrangements occur on one L chain allele at atime until a functional L chain is produced, after which the L chainpolypeptides can associate with the H chain homodimers to form a fullyfunctional B cell receptor for antigen (BCR).

The genes encoding various canine (e.g., the domestic dog and wolf) andmouse immunoglobulins have been extensively characterized, although thesequence and annotation of the canine Ig loci in the genome databases isnot yet complete. Priat, et al., describe whole-genome radiation mappingof the dog genome in Genomics, 54:361-78 (1998), and Bao, et al.,describe the molecular characterization of the V_(H) repertoire in Canisfamiliaris in Veterinary Immunology and Immunopathology, 137:64-75(2010). Blankenstein and Krawinkel describe the mouse variable heavychain region in Eur. J. Immunol., 17:1351-1357 (1987). The generation oftransgenic animals—such as mice having varied immunoglobulin loci—hasallowed the use of such transgenic animals in various research anddevelopment applications, e.g., in drug discovery and basic researchinto various biological systems. For example, the generation oftransgenic mice bearing human immunoglobulin genes is described inInternational Application WO 90/10077 and WO 90/04036. WO 90/04036describes a transgenic mouse with an integrated human immunoglobulin“mini” locus. WO 90/10077 describes a vector containing theimmunoglobulin dominant control region for use in generating transgenicanimals.

Numerous methods have been developed for modifying the mouse endogenousimmunoglobulin variable region gene locus with, e.g., humanimmunoglobulin sequences to create partly or fully-human antibodies fordrug discovery purposes. Examples of such mice include those describedin, e.g., U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986;6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010;5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016;5,612,205; and 5,591,669. However, many of the fully humanizedimmunoglobulin transgenic mice exhibit suboptimal antibody productionbecause B cell development in these mice is severely hampered byinefficient V(D)J recombination, and by inability of the fully humanantibodies/BCRs to function optimally with mouse signaling proteins.Other humanized immunoglobulin transgenic mice, in which the mousecoding sequence have been “swapped” with human sequences, are very timeconsuming and expensive to create due to the approach of replacingindividual mouse exons with the syntenic human counterpart.

The use of antibodies that function as drugs is not necessarily limitedto the prevention or therapy of human disease. Companion animals such asdogs suffer from some of the same afflictions as humans, e.g., cancer,atopic dermatitis and chronic pain. Monoclonal antibodies targetingCD20, IgE and Nerve Growth Factor, respectively, are already inveterinary use as for treatment of these conditions. However, beforeclinical use these monoclonal antibodies, which were made in mice, hadto be caninized, i.e., their amino acid sequence had to be changed frommouse to dog, in order to prevent an immune response in the recipientdogs. Based on the foregoing, it is clear that a need exists forefficient and cost-effective methods to produce canine antibodies forthe treatment of diseases in dogs. More particularly, there is a need inthe art for small, rapidly breeding, non-canine mammals capable ofproducing antigen-specific canine immunoglobulins. Such non-caninemammals are useful for generating hybridomas capable of large-scaleproduction of canine monoclonal antibodies.

In accordance with the foregoing object, transgenic nonhuman animals areprovided which are capable of producing an antibody with canine Vregions.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present invention comprises a non-canine mammalian cell and anon-canine mammal having a genome comprising an exogenously introducedpartly canine immunoglobulin locus, where the introduced locus comprisescoding sequences of the canine immunoglobulin variable region genes andnon-coding sequences based on the endogenous immunoglobulin variableregion locus of the non-canine mammalian host. Thus, the non-caninemammalian cell or mammal of the invention is capable of expressing achimeric B cell receptor (BCR) or antibody comprising H and L chainvariable regions that are fully canine in conjunction with therespective constant regions that are native to the non-canine mammalianhost cell or mammal. Preferably, the transgenic cells and animals of theinvention have genomes in which part or all of the endogenousimmunoglobulin variable region gene locus is removed.

At a minimum, the production of chimeric canine monoclonal antibodies ina non-canine mammalian host requires the host to have at least one locusthat expresses chimeric canine immunoglobulin H or L chain. In mostaspects, there are one heavy chain locus and two light chain loci that,respectively, express chimeric canine immunoglobulin H and L chains.

In some aspects, the partly canine immunoglobulin locus comprises canineV_(H) coding sequences and non-coding regulatory or scaffold sequencespresent in the endogenous V_(H) gene locus of the non-canine mammalianhost. In these aspects, the partly canine immunoglobulin locus furthercomprises canine D_(H) and J_(H) gene segment coding sequences inconjunction with the non-coding regulatory or scaffold sequences presentin the vicinity of the endogenous D_(H) and J_(H) gene segments of thenon-canine mammalian host cell genome.

In other aspects, the partly canine immunoglobulin locus comprisescanine V_(L) coding sequences and non-coding regulatory or scaffoldsequences present in the endogenous V_(L) gene locus of the non-caninemammalian host. More preferably, the exogenously introduced, partlycanine immunoglobulin locus comprising canine V_(L) coding sequencesfurther comprises canine L-chain J gene segment coding sequences andnon-coding regulatory or scaffold sequences present in the vicinity ofthe endogenous L-chain J gene segments of the non-canine mammalian hostcell genome.

In certain aspects, the non-canine mammal is a rodent, preferably amouse or rat.

In one specific aspect, the invention provides a method for generating anon-canine mammalian cell comprising a partly canine immunoglobulinlocus, said method comprising: a) introducing two or more recombinasetargeting sites into the genome of a non-canine mammalian host cell andintegrating at least one site upstream and at least one site downstreamof a genomic region comprising endogenous immunoglobulin variable regiongenes; and b) introducing into the non-canine mammalian host cell viarecombinase-mediated cassette exchange (RMCE) an engineered partlycanine immunoglobulin variable gene locus comprising canineimmunoglobulin variable region gene coding sequences and non-codingregulatory or scaffold sequences corresponding to the non-codingregulatory or scaffold sequences present in the endogenousimmunoglobulin variable region gene locus of the non-canine mammalianhost.

In another aspect, the method further comprises deleting the genomicregion flanked by the two exogenously introduced recombinase targetingsites prior to step b.

In a specific aspect of this method, the exogenously introduced,engineered partly canine immunoglobulin locus comprises canine V_(H)gene segment coding sequences, and further comprises i) canine D_(H) andJ_(H) gene segment coding sequences and ii) non-coding regulatory orscaffold sequences upstream of the canine D_(H) gene segments (pre-Dsequences, FIG. 1) that correspond to the sequences present upstream ofthe endogenous D_(H) gene segments in the genome of the non-caninemammalian host. Furthermore, these upstream scaffold sequences maycontain non-immunoglobulin genes, such as ADAM6 (FIG. 1) needed for malefertility (Nishimura et al. Developmental Biol. 233(1): 204-213 (2011)).The partly canine immunoglobulin locus is introduced into the host cellusing recombinase targeting sites that have been previously introducedupstream of the endogenous immunoglobulin V_(H) gene locus anddownstream of the endogenous J_(H) gene locus on the same chromosome.

In other aspects, the non-coding regulatory or scaffold sequences derive(at least partially) from other sources, e.g., they could be rationallyor otherwise designed sequences, sequences that are a combination ofcanine and other designed sequences, or sequences from other species.

In yet another specific aspect of the method, the introduced engineeredpartly canine immunoglobulin locus comprises canine immunoglobulin V_(L)gene segment coding sequences, and further comprises i) canine L-chain Jgene segment coding sequences and ii) non-coding regulatory or scaffoldsequences corresponding to the non-coding regulatory or scaffoldsequences present in the endogenous L chain locus of the non-caninemammalian host cell genome. The engineered partly canine immunoglobulinlocus is preferably introduced into the host cell using recombinasetargeting sites that have been previously introduced upstream of theendogenous immunoglobulin V_(L) gene locus and downstream of theendogenous J gene locus on the same chromosome.

Preferably, the engineered partly canine immunoglobulin locus issynthesized as a single nucleic acid, and introduced into the non-caninemammalian host cell as a single nucleic acid region. The engineeredpartly canine immunoglobulin locus may also be synthesized in two ormore contiguous segments, and introduced to the mammalian host cell asdiscrete segments. The engineered partly canine immunoglobulin locus canalso be produced using recombinant methods and isolated prior beingintroduced into the non-canine mammalian host cell.

In another aspect, the invention provides methods for generating anon-canine mammalian cell comprising an engineered partly canineimmunoglobulin locus, said method comprising: a) introducing into thegenome of a non-canine mammalian host cell two or more sequence-specificrecombination sites that are not capable of recombining with oneanother, wherein at least one recombination site is introduced upstreamof an endogenous immunoglobulin variable region gene locus while atleast one recombination site is introduced downstream of the endogenousimmunoglobulin variable region gene locus on the same chromosome; b)providing a vector comprising an engineered partly canine immunoglobulinlocus having i) canine immunoglobulin variable region gene codingsequences and ii) non-coding regulatory or scaffold sequences based onan endogenous immunoglobulin variable region gene locus of the host cellgenome, wherein the partly canine immunoglobulin locus is flanked by thesame two sequence-specific recombination sites that flank the endogenousimmunoglobulin variable region gene locus of the host cell of a); c)introducing into the host cell the vector of step b) and a site specificrecombinase capable of recognizing the two recombinase sites; d)allowing a recombination event to occur between the genome of the cellof a) and the engineered partly canine immunoglobulin locus, resultingin a replacement of the endogenous immunoglobulin variable region genelocus with the engineered partly canine immunoglobulin variable regiongene locus. In a specific aspect of this method, the partly canineimmunoglobulin locus comprises V_(H) immunoglobulin gene segment codingsequences, and further comprises i) canine D_(H) and J_(H) gene segmentcoding sequences, ii) non-coding regulatory or scaffold sequencessurrounding the codons of individual V_(H), D_(H), and J_(H) genesegments present endogenously in the genome of the non-canine mammalianhost, and iii) pre-D sequences based on the endogenous genome of thenon-canine mammalian host cell. The recombinase targeting sites areintroduced upstream of the endogenous immunoglobulin V_(H) gene locusand downstream of the endogenous D_(H) and J_(H) gene locus.

Thus, in some embodiments, there is provided a transgenic rodent with agenome deleted of a rodent endogenous immunoglobulin variable gene locusand into which the deleted rodent endogenous immunoglobulin variablegene locus has been replaced with an engineered partly canineimmunoglobulin locus comprising canine immunoglobulin variable genecoding sequences and non-coding regulatory or scaffold sequences basedon the rodent endogenous immunoglobulin variable gene locus, wherein theengineered partly canine immunoglobulin locus of the transgenic rodentis functional and expresses immunoglobulin chains comprised of caninevariable domains and rodent constant domains. In some aspects, theengineered partly canine immunoglobulin locus comprises canine V_(H),D_(H), and J_(H) coding sequences, and in some aspects, the engineeredpartly canine immunoglobulin locus comprises canine V_(L) and J_(L)coding sequences. Some aspects provide a cell of B lymphocyte lineagefrom the transgenic rodent, a part or whole immunoglobulin moleculecomprising canine variable domains and rodent constant domains derivedfrom the cell of B lymphocyte lineage, a hybridoma cell derived from thecell of B lymphocyte, a part or whole immunoglobulin molecule comprisingcanine variable domains and rodent constant domains derived from thehybridoma cell, an immortalized cell derived from the cell of Blymphocyte lineage, a part or whole immunoglobulin molecule comprisingcanine variable domains and rodent constant domains derived from theimmortalized cell. Other aspects of the invention provide a transgenicrodent, wherein the engineered partly canine immunoglobulin locuscomprises canine V_(L) and J_(L) coding sequences, and a transgenicrodent, wherein the engineered partly canine immunoglobulin locicomprise canine V_(H), D_(H), and J_(H) or V_(L) and J_(L) codingsequences. In some aspects, the rodent is a mouse. In some aspects, thenon-coding regulatory sequences comprise the following sequences ofedogenous host origin: promoters preceding each V gene segment, splicesites, and recombination signal sequences for V(D)J recombination; inother aspects, the engineered partly canine immunoglobulin locus furthercomprises one or more of the following sequences of endogenous hostorigin: ADAM6 gene, a Pax-5-Activated Intergenic Repeat (PAIR) elements,or CTCF binding sites from a heavy chain intergenic control region 1.

Preferably, the non-canine mammalian cell for use in each of the abovemethods is a mammalian cell, and more preferably a mammalian embryonicstem (ES) cell.

Once the cells have been subjected to the replacement of the endogenousimmunoglobulin variable region gene locus by the introduced partlycanine immunoglobulin variable region gene locus, the cells are selectedand preferably isolated. In a preferred aspect of the invention, thecells are non-canine mammalian ES cells, preferably rodent ES cells, andat least one isolated ES cell clone is then utilized to create atransgenic non-canine mammal expressing the engineered partly canineimmunoglobulin variable region gene locus.

An embodiment of the invention provides a method for generating thetransgenic rodent, said method comprising: a) integrating at least onetarget site for a site-specific recombinase in a rodent cell's genomeupstream of an endogenous immunoglobulin variable gene locus and atleast one target site for a site-specific recombinase downstream of theendogenous immunoglobulin variable gene locus, wherein the endogenousimmunoglobulin variable locus comprises V_(H), D_(H) and J_(H) genesegments, or Vκ and Jκ gene segments, or Vλ and Jλ gene segments, or Vλ,Jλ and Cλ gene segments; b) providing a vector comprising an engineeredpartly canine immunoglobulin locus, said engineered partly canineimmunoglobulin locus comprising chimeric canine immunoglobulin genesegments, wherein each of the partly canine immunoglobulin gene segmentcomprises canine immunoglobulin variable gene coding sequences androdent non-coding regulatory or scaffold sequences, with the partlycanine immunoglobulin variable gene locus being flanked by target sitesfor a site-specific recombinase wherein the target sites are capable ofrecombining with the target sites introduced into the rodent cell; c)introducing into the cell the vector and a site-specific recombinasecapable of recognizing the target sites; d) allowing a recombinationevent to occur between the genome of the cell and the engineered partlycanine immunoglobulin locus resulting in a replacement of the endogenousimmunoglobulin variable gene locus with the engineered partly canineimmunoglobulin locus; e) selecting a cell that comprises the engineeredpartly canine immunoglobulin variable locus generated in step d); andutilizing the cell to create a transgenic rodent comprising partlycanine the engineered partly canine immunoglobulin variable locus. Insome aspects, the cell is a rodent embryonic stem (ES) cell, and in someaspects the cell is a mouse embryonic stem (ES) cell. Some aspects ofthis method further comprise after, after step a) and before step b), astep of deleting the endogenous immunoglobulin variable gene locus byintroduction of a recombinase that recognizes a first set of targetsites, wherein the deleting step leaves in place at least one set oftarget sites that are not capable of recombining with one another in therodent cell's genome. In some aspects, the vector comprises canineV_(H), D_(H), and J_(H), coding sequences, and in some aspects thevector comprises canine V_(L) and J_(L) coding sequences. In someaspects, the vector further comprises a promoter, splice sites, andrecombination signal sequences.

In another aspect, the invention provides a method for generating atransgenic non-canine mammal comprising an exogenously introduced,engineered partly canine immunoglobulin variable region gene locus, saidmethod comprising: a) introducing into the genome of a non-caninemammalian host cell one or more sequence-specific recombination sitesthat flank an endogenous immunoglobulin variable region gene locus andare not capable of recombining with one another; b) providing a vectorcomprising a partly canine immunoglobulin locus having i) caninevariable region gene coding sequences and ii) non-coding regulatory orscaffold sequences based on the endogenous host immunoglobulin variableregion gene locus, wherein the coding and non-coding regulatory orscaffold sequences are flanked by the same sequence-specificrecombination sites as those introduced to the genome of the host cellof a); c) introducing into the cell the vector of step b) and asite-specific recombinase capable of recognizing one set of recombinasesites; d) allowing a recombination event to occur between the genome ofthe cell of a) and the engineered partly canine immunoglobulin variableregion gene locus, resulting in a replacement of the endogenousimmunoglobulin variable region gene locus with the partly canineimmunoglobulin locus; e) selecting a cell which comprises the partlycanine immunoglobulin locus; and f) utilizing the cell to create atransgenic animal comprising the partly canine immunoglobulin locus.

In a specific aspect, the engineered partly canine immunoglobulin locuscomprises canine V_(H), D_(H), and J_(H) gene segment coding sequences,and non-coding regulatory and scaffold pre-D sequences (including afertility-enabling gene) present in the endogenous genome of thenon-canine mammalian host. The sequence-specific recombination sites arethen introduced upstream of the endogenous immunoglobulin V_(H) genesegments and downstream of the endogenous J_(H) gene segments.

The invention provides another method for generating a transgenicnon-canine animal comprising an engineered partly canine immunoglobulinlocus, said method comprising: a) providing a non-canine mammalian cellhaving a genome that comprises two sets of sequence-specificrecombination sites that are not capable of recombining with oneanother, and which flank a portion of an endogenous immunoglobulinvariable region gene locus of the host genome; b) deleting the portionof the endogenous immunoglobulin locus of the host genome byintroduction of a recombinase that recognizes a first set ofsequence-specific recombination sites, wherein such deletion in thegenome retains a second set of sequence-specific recombination sites; c)providing a vector comprising an engineered partly canine immunoglobulinvariable region gene locus having canine coding sequences and non-codingregulatory or scaffold sequences based on the endogenous immunoglobulinvariable region gene locus, where the coding and non-coding regulatoryor scaffold sequences are flanked by the second set of sequence-specificrecombination sites; d) introducing the vector of step c) and asite-specific recombinase capable of recognizing the second set ofsequence-specific recombination sites into the cell; e) allowing arecombination event to occur between the genome of the cell and thepartly canine immunoglobulin locus, resulting in a replacement of theendogenous immunoglobulin locus with the engineered partly canineimmunoglobulin variable locus; f) selecting a cell that comprises thepartly canine immunoglobulin variable region gene locus; and g)utilizing the cell to create a transgenic animal comprising theengineered partly canine immunoglobulin variable region gene locus.

The invention provides yet another method for generating a transgenicnon-canine mammal comprising an engineered partly canine immunoglobulinlocus, said method comprising: a) providing a non-canine mammalianembryonic stem ES cell having a genome that contains twosequence-specific recombination sites that are not capable ofrecombining with each other, and which flank the endogenousimmunoglobulin variable region gene locus; b) providing a vectorcomprising an engineered partly canine immunoglobulin locus comprisingcanine immunoglobulin variable gene coding sequences and non-codingregulatory or scaffold sequences based on the endogenous immunoglobulinvariable region gene locus, where the partly canine immunoglobulin locusis flanked by the same two sequence-specific recombination sites thatflank the endogenous immunoglobulin variable region gene locus in the EScell; c) bringing the ES cell and the vector into contact with asite-specific recombinase capable of recognizing the two recombinasesites under appropriate conditions to promote a recombination eventresulting in the replacement of the endogenous immunoglobulin variableregion gene locus with the engineered partly canine immunoglobulinvariable region gene locus in the ES cell; d) selecting an ES cell thatcomprises the engineered partly canine immunoglobulin locus; and e)utilizing the cell to create a transgenic animal comprising theengineered partly canine immunoglobulin locus.

In a specific aspect of the invention, the transgenic non-canine mammalis a rodent, e.g., a mouse or a rat.

The invention further provides a non-canine mammalian cell and anon-canine transgenic mammal expressing an introduced immunoglobulinvariable region gene locus having canine variable region gene codingsequences and non-coding regulatory or scaffold sequences based on theendogenous non-canine immunoglobulin locus of the host genome, where thenon-canine mammalian cell and transgenic animal express chimericantibodies consisting of fully canine H and/or L chain variable domainsin conjunction with their respective constant regions that are native tothe non-canine mammalian cell or animal.

Further, the invention also provides B cells from transgenic animalsthat are capable of expressing partly canine antibodies having fullycanine variable sequences, wherein such B cells are immortalized toprovide a source of a monoclonal antibody specific for a particularantigen.

The invention additionally provides canine immunoglobulin variableregion gene sequences cloned from B cells for use in the productionand/or optimization of antibodies for diagnostic, preventative andtherapeutic uses.

Also, the invention provides hybridoma cells that are capable ofproducing partly canine monoclonal antibodies having fully canineimmunoglobulin variable region sequences.

The invention also provides methods for removing the V_(H) and V_(L)exons that encode the H and L chain immunoglobulin variable domains fromthe monoclonal antibody-producing hybridomas and reconfigure them tocontain canine constant regions, thereby creating a fully canineantibody that is not immunogenic when injected into dogs.

These and other aspects, objects and features are described in moredetail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the endogenous mouse Igh locus locatedat the telomeric end of chromosome 12.

FIG. 2 is a schematic diagram illustrating the strategy of targeting byhomologous recombination to introduce a first set of sequence-specificrecombination sites into a region upstream of the H chain variableregion gene locus in the genome of a non-canine mammalian host cell.

FIG. 3 is another schematic diagram illustrating the strategy oftargeting by homologous recombination to introduce a first set ofsequence-specific recombination sites into a region upstream of the Hchain variable region gene locus in the genome of a non-canine mammalianhost cell.

FIG. 4 is a schematic diagram illustrating the introduction of a secondset of sequence-specific recombination sites into a region downstream ofthe H chain variable region gene locus in the genome of a non-caninemammalian cell via a homology targeting vector.

FIG. 5 is a schematic diagram illustrating deletion of the endogenousimmunoglobulin H chain variable region gene locus from the genome of thenon-canine mammalian host cell.

FIG. 6 is a schematic diagram illustrating the RMCE strategy tointroduce an engineered partly canine immunoglobulin H chain locus intothe non-canine mammalian host cell genome that has been previouslymodified to delete the endogenous immunoglobulin H chain variable regiongene locus.

FIG. 7 is a schematic diagram illustrating the RMCE strategy tointroduce an engineered partly canine immunoglobulin H chain locuscomprising additional regulatory sequences into the non-canine mammalianhost cell genome that has been previously modified to delete theendogenous immunoglobulin H chain variable region genes.

FIG. 8 is a schematic diagram illustrating the introduction of anengineered partly canine immunoglobulin H chain variable region genelocus into the endogenous immunoglobulin H chain locus of the mousegenome.

FIG. 9 is a schematic diagram illustrating the introduction of anengineered partly canine immunoglobulin κL chain variable region genelocus into the endogenous immunoglobulin κL chain locus of the mousegenome.

FIG. 10 is a schematic diagram illustrating the introduction of anengineered partly canine immunoglobulin λL chain variable region genelocus into the endogenous immunoglobulin λL chain locus of the mousegenome.

FIG. 11 is a schematic diagram illustrating the introduction of anengineered partly canine immunoglobulin locus comprising a canine V_(H)minilocus via RMCE.

DEFINITIONS

The terms used herein are intended to have the plain and ordinarymeaning as understood by those of ordinary skill in the art. hefollowing definitions are intended to aid the reader in understandingthe present invention, but are not intended to vary or otherwise limitthe meaning of such terms unless specifically indicated.

The term “locus” as used herein refers to a chromosomal segment ornucleic acid sequence that, respectively, is present endogenously in thegenome or is (or about to be) exogenously introduced into the genome.For example, an immunoglobulin locus may include part or all of thegenes (i.e., V, D, J gene segments as well as constant region genes) andintervening sequences (i.e., introns, enhancers, etc.) supporting theexpression of immunoglobulin H or L chain polypeptides. Thus, a locus(e.g., V_(H) gene locus) may refer to a specific portion of a largerlocus (e.g., immunoglobulin H chain locus).

The term “immunoglobulin variable region gene” as used herein refers toa V, D, or J gene segment that encodes a portion of an immunoglobulin Hor L chain variable domain. The term “immunoglobulin variable regiongene locus” as used herein refers to part of, or the entire, chromosomalsegment or nucleic acid strand containing clusters of the V, D, or Jgene segments and may include the non-coding regulatory or scaffoldsequences.

“Partly canine” as used herein refers to a strand of nucleic acids, ortheir expressed protein and RNA products, comprising sequencescorresponding to the sequences found in a given locus of both a canineand a non-canine mammalian host. “Partly canine” as used herein alsorefers to an animal comprising nucleic acid sequences from both a canineand a non-canine mammal, preferably a rodent. In the context of partlycanine sequences of the invention, the partly canine nucleic acids havecoding sequences of canine immunoglobulin H or L chain variable regiongene segments and sequences based on the non-coding regulatory orscaffold sequences of the endogenous immunoglobulin locus of thenon-canine mammal. The term “based on” when used with reference toendogenous non-coding regulatory or scaffold sequences from a non-caninemammalian host cell genome refers to the non-coding regulatory orscaffold sequences that are present in the corresponding endogenouslocus of the mammalian host cell genome. “Non-coding regulatorysequences” refer to sequences that are known to be essential for (i)V(D)J recombination, (ii) isotype switching, and (iii) proper expressionof the full-length immunoglobulin H or L chains following V(D)Jrecombination. “Non-coding regulatory sequences” may further include thefollowing sequences of endogenous origin: enhancer and locus controlelements such as the CTCF and PAIR sequences (Proudhon, et al., Adv.Immunol. 128:123-182 (2015)); promoters preceding each endogenous V genesegment; splice sites; introns; recombination signal sequences flankingeach V, D, or J gene segment. Preferably, the “non-coding regulatorysequences” of the partly canine immunoglobulin locus share at least 70%homology with the corresponding non-coding sequences found in thetargeted endogenous immunoglobulin locus of the non-canine mammalianhost cell. “Scaffold sequences” refer to non-immunoglobulin genes, suchas ADAM6, and other sequences with unknown functions present in theendogenous immunoglobulin locus of the host cell genome. In certainaspects, the non-coding regulatory or scaffold sequences are derived (atleast partially) from other sources—e.g., they could be rationally orotherwise designed sequences, sequences that are a combination of canineand other designed sequences, or sequences from other species. It is tobe understood that the phrase “non-coding regulatory or scaffoldsequence” is inclusive in meaning (i.e., referring to both thenon-coding regulatory sequence and the scaffold sequence existing in agiven locus).

The term “homology targeting vector” refers to a nucleic acid sequenceused to modify the endogenous genome of a mammalian host cell byhomologous recombination; such nucleic acid sequence may comprise (i)targeting sequences with significant homologies to the correspondingendogenous sequences flanking a locus to be modified that is present inthe genome of the non-canine mammalian host, (ii) at least onesequence-specific recombination site, (iii) non-coding regulatory orscaffold sequences, and (iv) optionally one or more selectable markergenes. As such, a homology targeting vector can be used in the presentinvention to introduce a sequence-specific recombination site intoparticular region of a host cell genome.

“Site-specific recombination” or “sequence-specific recombination”refers to a process of DNA rearrangement between two compatiblerecombination sequences (also referred to as “sequence-specificrecombination sites” or “site-specific recombination sequences”)including any of the following three events: a) deletion of apreselected nucleic acid flanked by the recombination sites; b)inversion of the nucleotide sequence of a preselected nucleic acidflanked by the recombination sites, and c) reciprocal exchange ofnucleic acid sequences proximate to recombination sites located ondifferent nucleic acid strands. It is to be understood that thisreciprocal exchange of nucleic acid segments can be exploited as atargeting strategy to introduce an exogenous nucleic acid sequence intothe genome of a host cell.

The term “targeting sequence” refers to a sequence homologous to DNAsequences in the genome of a cell that flank or are adjacent to theregion of an immunoglobulin locus to be modified. The flanking oradjacent sequence may be within the locus itself or upstream ordownstream of coding sequences in the genome of the host cell. Targetingsequences are inserted into recombinant DNA vectors which are used totransfect, e.g., ES cells such that sequences to be inserted into thehost cell genome, such as the sequence of a recombination site, areflanked by the targeting sequences of the vector.

The term “site-specific targeting vector” as used herein refers to avector comprising a nucleic acid encoding a sequence-specificrecombination site, an engineered partly canine locus, and optionally aselectable marker gene, which is used to modify an endogenousimmunoglobulin locus in a host using recombinase-mediated site-specificrecombination. The recombination site of the targeting vector issuitable for site-specific recombination with another correspondingrecombination site that has been inserted into a genomic sequence of thehost cell (e.g., via a homology targeting vector), adjacent to animmunoglobulin locus that is to be modified. Integration of anengineered partly canine sequence into a recombination site in animmunoglobulin locus results in replacement of the endogenous locus bythe exogenously introduced partly canine region.

The term “transgene” is used herein to describe genetic material thathas been or is about to be artificially inserted into the genome of acell, and particularly a cell of a mammalian host animal. The term“transgene” as used herein refers to a partly canine nucleic acid, e.g.,a partly canine nucleic acid in the form of an engineered expressionconstruct and/or a targeting vector.

“Transgenic animal” refers to a non-canine animal, usually a mammal,having an exogenous nucleic acid sequence present as an extrachromosomalelement in a portion of its cells or stably integrated into its germline DNA (i.e., in the genomic sequence of most or all of its cells). Inthe present invention, a partly canine nucleic acid is introduced intothe germ line of such transgenic animals by genetic manipulation of, forexample, embryos or embryonic stem cells of the host animal according tomethods well known in the art.

A “vector” includes plasmids and viruses and any DNA or RNA molecule,whether self-replicating or not, which can be used to transform ortransfect a cell.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and sequencing technology,which are within the skill of those who practice in the art. Suchconventional techniques include polymer array synthesis, hybridizationand ligation of polynucleotides, and detection of hybridization using alabel. Specific illustrations of suitable techniques can be had byreference to the examples herein. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGreen, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series(Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation:A Laboratory Manual; Dieffenbach and Veksler, Eds. (2007), PCR Primer: ALaboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: AMolecular Cloning Manual; Mount (2004), Bioinformatics: Sequence andGenome Analysis; Sambrook and Russell (2006), Condensed Protocols fromMolecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002),Molecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H.Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A PracticalApproach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger,Principles of Biochemistry 3.sup.rd Ed., W. H. Freeman Pub., New York,N.Y.; and Berg et al. (2002) Biochemistry, 5.sup.th Ed., W.H. FreemanPub., New York, N.Y., all of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a locus” refers toone or more loci, and reference to “the method” includes reference toequivalent steps and methods known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

In the humoral immune system, a diverse antibody repertoire is producedby combinatorial and junctional diversity of IgH (Igh) and IgL chain(Igl) gene loci by a process termed V(D)J recombination. In thedeveloping B cell, the first recombination event to occur is between oneD and one J gene segment of the heavy chain locus, and the DNA betweenthese two gene segments is deleted. This D-J recombination is followedby the joining of one V gene segment from a region upstream of the newlyformed DJ complex, forming a rearranged VDJ exon. All other sequencesbetween the recombined V and D gene segments of the newly generated VDJexon are deleted from the genome of the individual B cell. Thisrearranged exon is ultimately expressed on the B cell surface as thevariable region of the H-chain polypeptide, which is associated with anL-chain polypeptide to form the B cell receptor (BCR). The murine andcanine Ig loci are highly complex in the numbers of features theycontain and in how their coding regions are diversified by V(D)Jrearrangement; however, this complexity does not extend to the basicdetails of the structure of each variable region gene segment. The V, Dand J gene segments are highly uniform in their compositions andorganizations. For example, V gene segments have the following featuresthat are arranged in essentially invariant sequential fashion inimmunoglobulin loci: a short transcriptional promoter region (<600 bp inlength), an exon encoding the majority of the signal peptide for theantibody chain; an intron; an exon encoding a small part of the signalpeptide of the antibody chain and the majority of the antibody variabledomain, and a 3′ recombination signal sequence necessary for V(D)Jrearrangement. Similarly, D gene segments have the following necessaryand invariant features: a 5′ recombination signal sequence, a codingregion and a 3′ recombination signal sequence. The J gene segments havethe following necessary and invariant features: a 5′ recombinationsignal sequence, a coding region and a 3′ splice donor sequence.

The present invention provides non-canine mammalian cells comprising anexogenously introduced, engineered partly canine nucleic acid sequencecomprising coding sequences for canine variable regions and non-codingregulatory or scaffold sequences present in the immunoglobulin locus ofthe mammalian host genome, e.g., mouse genomic non-coding sequences whenthe host mammal is a mouse. The canine genome V_(H) region comprisesapproximately 80 V_(H), 6 D_(H) and 3 J_(H) gene segments mapping to a1.28 Mb region of canine chromosome 8. The lambda coding region maps tocanine chromosome 26, while the kappa coding region maps to caninechromosome 17. The partly canine nucleic acid sequence allows thetransgenic animal to produce a heavy chain repertoire comprising canineV_(H) regions, while retaining the regulatory sequences and otherelements that can be found within the intervening sequences of the hostgenome (e.g., rodent) that help to promote efficient antibody productionand antigen recognition in the host. Similar to humans and mice, twotypes of Ig light chains (κ and λ) are expressed in dogs, though the κto λ ratio differs significantly among these animals. In mice,approximately 96% of light chains in the serum antibodies are the κtype, while the κ type in humans accounts for only 66% of the totalpopulation of Ig L chains. In contrast, the L chain repertoire in dogsis dominated by λ.

The present invention comprises the use of a synthetic, or recombinantlyproduced, partly canine nucleic acids engineered to comprise both caninecoding sequences and non-canine non-coding regulatory or scaffoldsequences from an immunoglobulin V_(H), Vλ or Vκ locus, or, in someaspects, a combination thereof.

In an aspect of the invention the synthetic H chain DNA segment containsthe ADAM6 gene needed for male fertility, Pax-5-Activated IntergenicRepeats (PAIR) elements involved in Igh locus contraction and CTCFbinding sites from the heavy chain intergenic control region 1, involvedin regulating normal VDJ rearrangement ((Proudhon, et al., Adv.Immunol., 128:123-182 (2015)), or various combinations thereof. Thelocations of these endogenous non-coding regulatory and scaffoldsequences in the mouse Igh locus are depicted in FIG. 1, whichillustrates from left to right: the ˜100 functional heavy chain variableregion gene segments (101); PAIR, Pax-5 Activated Intergenic Repeatsinvolved in Igh locus contraction for VDJ recombination (102); Adam6a, adisintegrin and metallopeptidase domain 6A gene required for malefertility (103); Pre-D region, a 21609 bp fragment upstream of the mostdistal D_(H) gene segment, Ighd-5 D_(H) (104); Intergenic Control Region1 (IGCR1) that contains CTCF insulator sites to regulate V_(H) genesegment usage (106); D_(H), diversity gene segments (10-15 depending onthe mouse strain) (105); four joining J_(H) gene segments (107); Eμ, theintronic enhancer involved in VDJ recombination (108); Sμ, the μ switchregion for isotype switching (109); eight heavy chain constant regiongenes: Cμ, Cδ, Cγ3, Cγ1, Cγ2b, C2γa/c, Cε, and Cα (110); 3′ RegulatoryRegion (3′RR) that controls isotype switching and somatic hypermutation(111). FIG. 1 is modified from a figure taken from Proudhon, et al.,Adv. Immunol., 128:123-182 (2015).

In preferred aspects of the invention, the engineered partly canineregion to be integrated into a mammalian host cell comprises all or asubstantial number of the known canine V_(H) gene segments. In someinstances, however, it may be desirable to use a subset of such V_(H)gene segments, and in specific instances even as few as one canine V_(H)coding sequence may be introduced into the cell or the animal of theinvention.

The preferred aspects of the invention comprise non-canine mammals andmammalian cells comprising an engineered partly canine immunoglobulinlocus that comprises coding sequences of canine V_(H), canine D_(H), andcanine J_(H) genes, and that further comprises non-coding regulatory andscaffold sequences, including pre-D sequences, based on the endogenousIgh locus of the non-canine mammalian host. In certain aspects, theexogenously introduced, engineered partly canine region can comprise afully recombined V(D)J exon.

In a specific aspect of the invention, the transgenic non-canine mammalis a rodent, preferably a mouse, comprising an exogenously introduced,engineered partly canine immunoglobulin locus comprising codons formultiple canine V_(H), canine D_(H), and canine J_(H) genes withintervening sequences, including a pre-D region, based on theintervening (non-coding regulatory or scaffold) sequences in the rodent.In a particularly preferred aspect, the transgenic non-canine rodentfurther comprises partly canine Igl loci comprising coding sequences ofcanine Vκ or Vλ genes, and Jκ or Jλ genes, respectively, in conjunctionwith their intervening (non-coding regulatory or scaffold) sequencescorresponding to the immunoglobulin intervening sequences present in theIgl loci of the rodent.

In an exemplary embodiment, as set forth in more detail in the Examplessection, the entire endogenous V_(H) immunoglobulin locus of the mousegenome is deleted and subsequently replaced with 80 canine V_(H) genesegments containing interspersed non-coding sequences corresponding tothe non-coding sequences of the J558 V_(H) locus of the mouse genome.The complete, exogenously introduced, engineered immunoglobulin locusfurther comprises canine D_(H) and J_(H) gene segments, as well as themouse pre-D region. Thus, the canine V_(H), D_(H), and J_(H) codonsequences are embedded in the rodent intergenic and intronic sequences.

The methods of the invention utilize a combination of homologousrecombination and site-specific recombination to create the cells andanimals of the invention. In some embodiments, a homology targetingvector is first used to introduce the sequence-specific recombinationsites into the mammalian host cell genome at a desired location in theendogenous immunoglobulin loci. Preferably, in the absence of arecombinase protein, the sequence-specific recombination site insertedinto the genome of a mammalian host cell by homologous recombinationdoes not affect expression and amino acid codons of any genes in themammalian host cell. This approach maintains the proper transcriptionand translation of the immunoglobulin genes which produce the desiredantibody after insertion of recombination sites and, optionally, anyadditional sequence such as a selectable marker gene. However, in somecases it is possible to insert a recombinase site and other sequencesinto an immunoglobulin locus sequence such that an amino acid sequenceof the antibody molecule is altered by the insertion, but the antibodystill retains sufficient functionality for the desired purpose. Examplesof such codon-altering homologous recombination may include theintroduction of polymorphisms into the endogenous locus and changing theconstant region exons so that a different isotype is expressed from theendogenous locus. The invention envisions encompassing such insertionsas well.

In specific aspects of the invention, the homology targeting vector canbe utilized to replace certain sequences within the endogenous genome aswell as to insert certain sequence-specific recombination sites and oneor more selectable marker genes into the host cell genome. It isunderstood by those of ordinary skill in the art that a selectablemarker gene as used herein can be exploited to weed out individual cellsthat have not undergone homologous recombination and cells that harborrandom integration of the targeting vector.

Exemplary methodologies for homologous recombination are described inU.S. Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992;and 5,464,764, each of which is incorporated by reference in itsentirety.

Site/Sequence-Specific Recombination

Site/sequence-specific recombination differs from general homologousrecombination in that short specific DNA sequences, which are requiredfor recognition by a recombinase, are the only sites at whichrecombination occurs. Depending on the orientations of these sites on aparticular DNA strand or chromosome, the specialized recombinases thatrecognize these specific sequences can catalyze i) DNA excision or ii)DNA inversion or rotation. Site-specific recombination can also occurbetween two DNA strands if these sites are not present on the samechromosome. A number of bacteriophage- and yeast-derived site-specificrecombination systems, each comprising a recombinase and specificcognate sites, have been shown to work in eukaryotic cells and aretherefore applicable for use in the present invention, and these includethe bacteriophage P1 Cre/lox, yeast FLP-FRT system, and the Dre systemof the tyrosine family of site-specific recombinases. Such systems andmethods of use are described, e.g. , in U.S. Pat. Nos. 7,422,889;7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and4,959,317, each of which is incorporated herein by reference to teachmethods of using such recombinases.

Other systems of the tyrosine family of site-specific recombinases suchas bacteriophage lambda integrase, HK2022 integrase, and in additionsystems belonging to the separate serine family of recombinases such asbacteriophage phiC31, R4Tp901 integrases are known to work in mammaliancells using their respective recombination sites, and are alsoapplicable for use in the present invention.

Since site-specific recombination can occur between two different DNAstrands, site-specific recombination occurrence can be utilized as amechanism to introduce an exogenous locus into a host cell genome by aprocess called recombinase-mediated cassette exchange (RMCE). The RMCEprocess can be exploited by the combined usage of wild-type and mutantsequence-specific recombination sites for the same recombinase proteintogether with negative selection. For example, a chromosomal locus to betargeted may be flanked by a wild-type LoxP site on one end and by amutant LoxP site on the other. Likewise, an exogenous vector containinga sequence to be inserted into the host cell genome may be similarlyflanked by a wild-type LoxP site on one end and by a mutant LoxP site onthe other. When this exogenous vector is transfected into the host cellin the presence of Cre recombinase, Cre recombinase will catalyze RMCEbetween the two DNA strands, rather than the excision reaction on thesame DNA strands, because the wild-type LoxP and mutant LoxP sites oneach DNA strand are incompatible for recombination with each other.Thus, the LoxP site on one DNA strand will recombine with a LoxP site onthe other DNA strand; similarly, the mutated LoxP site on one DNA strandwill only recombine with a likewise mutated LoxP site on the other DNAstrand.

The methods of the invention preferably utilize combined variants of thesequence-specific recombination sites that are recognized by the samerecombinase for RMCE. Examples of such sequence-specific recombinationsite variants include those that contain a combination of invertedrepeats or those which comprise recombination sites having mutant spacersequences. For example, two classes of variant recombinase sites areavailable to engineer stable Cre-loxP integrative recombination. Bothexploit sequence mutations in the Cre recognition sequence, eitherwithin the 8 bp spacer region or the 13-bp inverted repeats. Spacermutants such as lox511 (Hoess, et al., Nucleic Acids Res, 14:2287-2300(1986)), lox5171 and lox2272 (Lee and Saito, Gene, 216:55-65 (1998)),m2, m3, m7, and m11 (Langer, et al., Nucleic Acids Res, 30:3067-3077(2002)) recombine readily with themselves but have a markedly reducedrate of recombination with the wild-type site. This class of mutants hasbeen exploited for DNA insertion by RMCE using non-interacting Cre-Loxrecombination sites and non-interacting FLP recombination sites (Baerand Bode, Curr Opin Biotechnol, 12:473-480 (2001); Albert, et al., PlantJ, 7:649-659 (1995); Seibler and Bode, Biochemistry, 36:1740-1747(1997); Schlake and Bode, Biochemistry, 33:12746-12751 (1994)).

Inverted repeat mutants represent the second class of variantrecombinase sites. For example, LoxP sites can contain altered bases inthe left inverted repeat (LE mutant) or the right inverted repeat (REmutant). An LE mutant, lox71, has 5 bp on the 5′ end of the leftinverted repeat that is changed from the wild type sequence to TACCG(Araki, et al, Nucleic Acids Res, 25:868-872 (1997)). Similarly, the REmutant, lox66, has the five 3′-most bases changed to CGGTA. Invertedrepeat mutants are used for integrating plasmid inserts into chromosomalDNA with the LE mutant designated as the “target” chromosomal loxP siteinto which the “donor” RE mutant recombines. Post-recombination, loxPsites are located in cis, flanking the inserted segment. The mechanismof recombination is such that post-recombination one loxP site is adouble mutant (containing both the LE and RE inverted repeat mutations)and the other is wild type (Lee and Sadowski, Prog Nucleic Acid Res MolBiol, 80:1-42 (2005); Lee and Sadowski, J Mol Biol, 326:397-412 (2003)).The double mutant is sufficiently different from the wild-type site thatit is unrecognized by Cre recombinase and the inserted segment is notexcised.

In certain aspects, sequence-specific recombination sites can beintroduced into introns, as opposed to coding nucleic acid regions orregulatory sequences. This avoids inadvertently disrupting anyregulatory sequences or coding regions necessary for proper antibodyexpression upon insertion of sequence-specific recombination sites intothe genome of the animal cell.

Introduction of the sequence-specific recombination sites may beachieved by conventional homologous recombination techniques. Suchtechniques are described in references such as e.g., Sambrook andRussell (2001) (Molecular cloning: a laboratory manual 3rd ed. (ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) and Nagy, A.(2003). (Manipulating the mouse embryo: a laboratory manual, 3rd ed.(Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). GeneticRecombination: Nucleic acid, Homology (biology), Homologousrecombination, Non-homologous end joining, DNA repair, Bacteria,Eukaryote, Meiosis, Adaptive immune system, V(D)J recombination byFrederic P. Miller, Agnes F. Vandome, and John McBrewster(Paperback—Dec. 23, 2009).

Specific recombination into the genome can be facilitated using vectorsdesigned for positive or negative selection as known in the art. Inorder to facilitate identification of cells that have undergone thereplacement reaction, an appropriate genetic marker system may beemployed and cells selected by, for example, use of a selection tissueculture medium. However, in order to ensure that the genome sequence issubstantially free of extraneous nucleic acid sequences at or adjacentto the two end points of the replacement interval, desirably the markersystem/gene can be removed following selection of the cells containingthe replaced nucleic acid.

In one preferred aspect of the methods of the present invention, cellsin which the replacement of all or part of the endogenous immunoglobulinlocus has taken place are negatively selected against upon exposure to atoxin or drug. For example, cells that retain expression of HSV-TK canbe selected against by using nucleoside analogues such as ganciclovir.In another aspect of the invention, cells comprising the deletion of theendogenous immunoglobulin locus may be positively selected for by use ofa marker gene, which can optionally be removed from the cells followingor as a result of the recombination event. A positive selection systemthat may be used is based on the use of two non-functional portions of amarker gene, such as HPRT, that are brought together through therecombination event. These two portions are brought into functionalassociation upon a successful replacement reaction being carried out andwherein the functionally reconstituted marker gene is flanked on eitherside by further sequence-specific recombination sites (which aredifferent from the sequence-specific recombination sites used for thereplacement reaction), such that the marker gene can be excised from thegenome, using an appropriate site-specific recombinase.

The recombinase may be provided as a purified protein, or as a proteinexpressed from a vector construct transiently transfected into the hostcell or stably integrated into the host cell genome. Alternatively, thecell may be used first to generate a transgenic animal, which then maybe crossed with an animal that expresses said recombinase.

Because the methods of the invention can take advantage of two or moresets of sequence-specific recombination sites within the engineeredgenome, multiple rounds of RMCE can be exploited to insert the partlycanine immunoglobulin variable region genes into a non-canine mammalianhost cell genome.

Although not yet routine for the insertion of large DNA segments, CRISPRtechnology is another method to introduce the chimeric canine Ig locus.

Generation of Transgenic Animals

In specific aspects, the invention provides methods for the creation oftransgenic animals, preferably rodents, and more preferably mice,comprising the introduced partly canine immunoglobulin locus.

In one aspect, the host cell utilized for replacement of the endogenousimmunoglobulin genes is an embryonic stem (ES) cell, which can then beutilized to create a transgenic mammal. Thus, in accordance with oneaspect, the methods of the invention further comprise: isolating anembryonic stem cell which comprises the introduced partly canineimmunoglobulin locus and using said ES cell to generate a transgenicanimal that contains the replaced partly canine immunoglobulin locus.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific embodiments withoutdeparting from the spirit or scope of the invention as broadlydescribed. The present embodiments are, therefore, to be considered inall respects as illustrative and not restrictive.

Efforts have been made to ensure accuracy with respect to terms andnumbers used (e.g., vectors, amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees centigrade,and pressure is at or near atmospheric.

The examples illustrate targeting by both a 5′ vector and a 3′ vectorthat flank a site of recombination and introduction of synthetic DNA. Itwill be apparent to one skilled in art upon reading the specificationthat the 5′ vector targeting can take place first followed by the 3′, orthe 3′ vector targeting can take place followed by the 5′ vector. Insome circumstances, targeting can be carried out simultaneously withdual detection mechanisms.

Example 1 Introduction of an Engineered Partly Canine ImmunoglobulinVariable Region Gene Locus into the Immunoglobulin H Chain VariableRegion Gene Locus of a Non-Canine Mammalian Host Cell Genome

An exemplary method illustrating the introduction of an engineeredpartly canine immunoglobulin locus into the genomic locus of anon-mammalian ES cell is illustrated in more detail in FIGS. 2-6. InFIG. 2, a homology targeting vector (201) is provided comprising apuromycin phosphotransferase-thymidine kinase fusion protein (puro-TK)(203) flanked by two different recombinase recognition sites (e.g., FRT(207) and loxP (205) for Flp and Cre, respectively) and two differentmutant sites (e.g., modified mutant FRT (209) and mutant loxP (211))that lack the ability to recombine with their respective wild-typecounterparts/sites (i.e., wild-type FRT (207) and wild-type loxP (205)).The targeting vector comprises a diphtheria toxin receptor (DTR) cDNA(217) for use in negative selection of cells containing the introducedconstruct in future steps. The targeting vector also optionallycomprises a visual marker such as a green fluorescent protein (GFP) (notshown). The regions 213 and 215 are homologous to the 5′ and 3′portions, respectively, of a contiguous region (229) in the endogenousnon-canine locus that is 5′ of the genomic region comprising theendogenous non-canine V_(H) gene segments (219). The homology targetingvector (201) is introduced (202) into the ES cell, which has animmunoglobulin locus (231) comprising endogenous V_(H) gene segments(219), the pre-D region (221), the D_(H) gene segments (223), J_(H) genesegments (225), and the immunoglobulin constant gene region genes (227).The site-specific recombination sequences and the DTR cDNA from thehomology targeting vector (201) are integrated (204) into the non-caninegenome at a site 5′ of the endogenous mouse V_(H) gene locus, resultingin the genomic structure illustrated at 233. The ES cells that do nothave the exogenous vector (201) integrated into their genome can beselected against (killed) by including puromycin in the culture medium;only the ES cells that have stably integrated the exogenous vector (201)into their genome and constitutively express the puro-TK gene areresistant to puromycin.

FIG. 3 illustrates effectively the same approach as FIG. 2, except thatan additional set of sequence-specific recombination sites is added,e.g., a Rox site (331) and a modified Rox site (335) for use with theDre recombinase. In FIG. 3, a homology targeting vector (301) isprovided comprising a puro-TK fusion protein (303) flanked by wild typerecombinase recognition sites for FRT (307), loxP (305), and Rox (331)and mutant sites for FRT (309) loxP (311) and Rox (333) recombinasesthat lack the ability to recombine with the wild-type sites 307, 305 and331, respectively. The targeting vector also comprises a diphtheriatoxin receptor (DTR) cDNA (317). The regions 313 and 315 are homologousto the 5′ and 3′ portions, respectively, of a contiguous region (329) inthe endogenous non-canine locus that is 5′ of the genomic regioncomprising the endogenous mouse V_(H) gene segments (319). The homologytargeting is introduced (302) into the mouse immunoglobulin locus (339),which comprises the endogenous V_(H) gene segments (319), the pre-Dregion (321), the D_(H) gene segments (323), J_(H) (325) gene segments,and the constant region genes (327) of the Igh locus. The site-specificrecombination sequences and the DTR cDNA (317) in the homology targetingvector (301) are integrated (304) into the mouse genome at a site 5′ ofthe endogenous mouse V_(H) gene locus, resulting in the genomicstructure illustrated at 333.

As illustrated in FIG. 4, a second homology targeting vector (401) isprovided comprising an optional hypoxanthine-guaninephosphoribosyltransferase (HPRT) gene (435) that can be used forpositive selection in HPRT-deficient ES cells; a neomycin resistancegene (437); recombinase recognition sites FRT (407) and loxP (405), forFlp and Cre, respectively, which have the ability to recombine with FRT(407) and loxP (405) sites previously integrated into the mouse genomefrom the first homology targeting vector. The previous homologytargeting vector also consists of mutant FRT site (409), mutant loxPsite (411), a puro-TK fusion protein (403), and a DTR cDNA at a site 5′of the endogenous mouse V_(H) gene locus (419). The regions 429 and 439are homologous to the 5′ and 3′ portions, respectively, of a contiguousregion (441) in the endogenous mouse non-canine locus that is downstreamof the endogenous J_(H) gene segments (425) and upstream of the constantregion genes (427). The homology targeting vector is introduced (402)into the modified mouse immunoglobulin locus (431), which comprises theendogenous V_(H) gene segments (419), the pre-D region (421), the D_(H)gene segments (423) the J_(H) gene segments (425), and the constantregion genes (427). The site-specific recombination sequences (407,405), the HPRT gene (435) and a neomycin resistance gene (437) of thehomology targeting vector are integrated (404) into the mouse genomeupstream of the endogenous mouse constant region genes (427), resultingin the genomic structure illustrated at 433.

Once the recombination sites are integrated into the mammalian host cellgenome, the endogenous region of the immunoglobulin domain is thensubjected to recombination by introducing one of the recombinasescorresponding to the sequence-specific recombination sites integratedinto the genome, e.g., either Flp or Cre. Illustrated in FIG. 5 is amodified Igh locus of the mammalian host cell genome comprising twointegrated DNA fragments. One fragment comprising mutant FRT site (509),mutant LoxP site (511), puro-TK gene (503), wild-type FRT site (507),and wild-type LoxP site (505), and DTR cDNA (517) is integrated upstreamof the V_(H) gene locus (519). The other DNA fragment comprising HPRTgene (535), neomycin resistance gene (537), wild-type FRT site (507),and wild-type LoxP site (505) is integrated downstream of the pre-D(521), D_(H) (523) and J_(H) (525) gene loci, but upstream of theconstant region genes (527). In the presence of Flp or Cre (502), allthe intervening sequences between the wild-type FRT or wild-type LoxPsites including the DTR gene (517), the endogenous Igh variable regiongene loci (519, 521, 525), and the HPRT (535) and neomycin resistance(537) genes are deleted, resulting in a genomic structure illustrated at539. The procedure depends on the second targeting having occurred onthe same chromosome rather than on its homolog (i.e., in cis rather thanin trans). If the targeting occurs in cis as intended in this invention,the cells are not sensitive to negative selection after Cre- orFlp-mediated recombination by diphtheria toxin introduced into themedia, because the DTR gene which causes sensitivity to diphtheria toxinshould be absent (deleted) from the host cell genome. Likewise, ES cellsthat harbor random integration of the first and/or second targetingvector(s) are rendered sensitive to diphtheria toxin by presence of theundeleted DTR gene.

ES cells that are insensitive to diphtheria toxin are then screened forthe deletion of the endogenous variable region gene loci. The primaryscreening method for the deleted endogenous immunoglobulin locus can becarried out by Southern blotting, or by polymerase chain reaction (PCR)followed by confirmation with a secondary screening technique such asSouthern blotting.

FIG. 6 illustrates introduction of the engineered partly canine sequenceinto a non-canine genome previously modified to delete part of theendogenous Igh locus that encodes the heavy chain variable regiondomains as well as all the intervening sequences between the V_(H) andJ_(H) gene locus. A site-specific targeting vector (629) comprisingpartly canine V_(H) gene locus (619), endogenous non-canine pre-D generegion (621), partly canine D_(H) gene locus (623), partly canine J_(H)gene locus (625), as well as flanking mutant FRT (609), mutant LoxP(611), wild-type FRT (607), and wild-type LoxP (605) sites is introduced(602) into the host cell. Specifically, the partly canine V_(H) locus(619) comprises 47 functional canine V_(H) coding sequences inconjunction with the intervening sequences based on the endogenousnon-canine genome sequences; the pre-D region (621) comprises a 21.6 kbmouse sequence with significant homology to the corresponding region ofthe endogenous canine Igh locus; the D_(H) gene locus (623) comprisescodons of 6 D_(H) gene segments embedded in the intervening sequencessurrounding the endogenous non-canine D_(H) gene segments; and the J_(H)gene locus (625) comprises codons of 5 canine J_(H) gene segmentsembedded in the intervening sequences based on the endogenous non-caninegenome. The Igh locus (601) of the host cell genome has been previouslymodified to delete all the V_(H), D_(H), and J_(H) gene segmentsincluding the intervening sequences as described in FIG. 5. As aconsequence of this modification, the endogenous non-canine host cellIgh locus (601) is left with a puro-TK fusion gene, which is flanked bya mutant FRT site (609) and a mutant LoxP site (611) upstream as well asa wild-type FRT (607) and a wild-type LoxP (605) downstream. Uponintroduction of the appropriate recombinase (604), the partly canineimmunoglobulin locus is integrated into the genome upstream of theendogenous non-canine constant region genes (627), resulting in thegenomic structure illustrated at 631.

The sequences of the canine V_(H), D_(H) and J_(H) gene segment codingregions are in Table 1.

Primary screening procedure for the introduction of the partly canineimmunoglobulin locus can be carried out by Southern blotting, or by PCRwith confirmation from secondary screening methods such as Southernblotting. The screening methods are designed to detect the presence ofthe inserted V_(H) and/or J_(H) gene loci, as well as all theintervening sequences.

Example 2 Introduction of an Engineered Partly Canine ImmunoglobulinVariable Region Gene Locus Comprising Additional Non-Coding Regulatoryor Scaffold Sequences into the Immunoglobulin H Chain Variable RegionGene Locus of a Non-Canine Mammalian Host Cell Genome

In certain aspects, the partly canine immunoglobulin locus comprises theelements as described in Example 1, but with additional non-codingregulatory or scaffold sequences e.g., sequences strategically added tointroduce additional regulatory sequences, to ensure the desired spacingwithin the introduced immunoglobulin locus, to ensure that certaincoding sequences are in adequate juxtaposition with other sequencesadjacent to the replaced immunoglobulin locus, and the like. FIG. 7illustrates the introduction of a second exemplary engineered partlycanine sequence to the modified non-canine genome as produced in FIGS.2-5 and described in Example 1 above.

FIG. 7 illustrates introduction of the engineered partly canine sequenceinto the mouse genome previously modified to delete part of theendogenous non-canine Igh locus that encodes the heavy chain variableregion domains as well as all the intervening sequences between theendogenous V_(H) and J_(H) gene loci. A site-specific targeting vector(731) comprising an engineered partly canine immunoglobulin locus to beinserted into the non-canine host genome is introduced (702) into thegenomic region (701). The site-specific targeting vector (731)comprising a partly canine V_(H) gene locus (719), mouse pre-D region(721), partly canine D_(H) gene locus (723), partly canine J_(H) genelocus (725), PAIR elements (741), as well as flanking mutant FRT (709),mutant LoxP (711) wild-type FRT (707) and wild-type LoxP (705) sites isintroduced (702) into the host cell. Specifically, the engineered partlycanine V_(H) gene locus (719) comprises 80 canine V_(H) gene segmentcoding regions in conjunction with intervening sequences based on theendogenous non-canine genome sequences; the pre-D region (721) comprisesa 21.6 kb non-canine sequence present upstream of the endogenousnon-canine genome; the D_(H) region (723) comprises codons of 6 canineD_(H) gene segments embedded in the intervening sequences surroundingthe endogenous non-canine D_(H) gene segments; and the J_(H) gene locus(725) comprises codons of 3 canine J_(H) gene segments embedded in theintervening sequences based on the endogenous non-canine genomesequences. The Igh locus (701) of the host cell genome has beenpreviously modified to delete all the V_(H), D_(H), and J_(H) genesegments including the intervening sequences as described in relation toFIG. 5. As a consequence of this modification, the endogenous non-canineIgh locus (701) is left with a puro-TK fusion gene (703), which isflanked by a mutant FRT site (709) and a mutant LoxP site (711) upstreamas well as a wild-type FRT (707) and a wild-type LoxP (705) downstream.Upon introduction of the appropriate recombinase (704), the engineeredpartly canine immunoglobulin locus is integrated into the genomeupstream of the endogenous mouse constant region genes (727), resultingin the genomic structure illustrated at 729.

The primary screening procedure for the introduction of the engineeredpartly canine immunoglobulin region can be carried out by Southernblotting, or by PCR with confirmations from secondary screening methodssuch as Southern blotting. The screening methods are designed to detectthe presence of the inserted V_(H) and/or J_(H) gene loci, as well asall the intervening sequences.

Example 3 Introduction of an Engineered Partly Canine ImmunoglobulinLocus into the Immunoglobulin Heavy Chain Gene Locus of a Mouse Genome

A method for replacing a portion of a mouse genome with an engineeredpartly canine immunoglobulin locus is illustrated in FIG. 8. This methoduses introduction of a first site-specific recombinase recognitionsequence into the mouse genome followed by the introduction of a secondsite-specific recombinase recognition sequence into the mouse genome.The two sites flank the entire clusters of endogenous mouse V_(H), D_(H)and J_(H) region gene segments. The flanked region is deleted using therelevant site-specific recombinase, as described herein.

The targeting vectors (803, 805) employed for introducing thesite-specific recombinase sequences on either side of the V_(H) (815),D_(H) (817) and J_(H) (819) gene segment clusters and upstream of theconstant region genes (821) in the wild-type mouse immunoglobulin locus(801) include an additional site-specific recombination sequence thathas been modified so that it is still recognized efficiently by therecombinase, but does not recombine with unmodified sites. This mutantmodified site (e.g., lox5171) is positioned in the targeting vector suchthat after deletion of the endogenous V_(H), D_(H) and J_(H) genesegments (802) it can be used for a second site-specific recombinationevent in which a non-native piece of DNA is moved into the modified Ighlocus by RMCE. In this example, the non-native DNA is a syntheticnucleic acid comprising both canine and non-canine sequences (809).

Two gene targeting vectors are constructed to accomplish the processjust outlined. One of the vectors (803) comprises mouse genomic DNAtaken from the 5′ end of the Igh locus, upstream of the most distalV_(H) gene segment. The other vector (805) comprises mouse genomic DNAtaken from within the locus downstream of the J_(H) gene segments.

The key features of the 5′ vector (803) in order from 5′ to 3′ are asfollows: a gene encoding the diphtheria toxin A (DTA) subunit undertranscriptional control of a modified herpes simplex virus type Ithymidine kinase gene promoter coupled to two mutant transcriptionalenhancers from the polyoma virus (823); 4.5 Kb of mouse genomic DNAmapping upstream of the most distal V_(H) gene segment in the Igh locus(825); a FRT recognition sequence for the Flp recombinase (827); a pieceof genomic DNA containing the mouse Polr2a gene promoter (829); atranslation initiation sequence (methionine codon embedded in a “Kozak”consensus sequence, 835)); a mutated loxP recognition sequence (lox5171)for the Cre recombinase (831); a transcriptiontermination/polyadenylation sequence (pA. 833); a loxP recognitionsequence for the Cre recombinase (837); a gene encoding a fusion proteincomprised of a protein conferring resistance to puromycin fused to atruncated form of the thymidine kinase (pu-TK) under transcriptionalcontrol of the promoter from the mouse phosphoglycerate kinase 1 gene(839); and 3 Kb of mouse genomic DNA (841) mapping close to the 4.5 Kbmouse genomic DNA sequence present near the 5′ end of the vector andarranged in the native relative orientation.

The key features of the 3′ vector (805) in order from 5′ to 3′ are asfollows; 3.7 Kb of mouse genomic DNA mapping within the intron betweenthe J_(H) and C_(H) gene loci (843); an HPRT gene under transcriptionalcontrol of the mouse Polr2a gene promoter (845); a neomycin resistancegene under the control of the mouse phosphoglycerate kinase 1 genepromoter (847); a loxP recognition sequence for the Cre recombinase(837); 2.1 Kb of mouse genomic DNA (849) that maps immediatelydownstream of the 3.7 Kb mouse genomic DNA fragment present near the 5′end of the vector and arranged in the native relative orientation; and agene encoding the DTA subunit under transcriptional control of amodified herpes simplex virus type I thymidine kinase gene promotercoupled to two mutant transcriptional enhancers from the polyoma virus(823).

Mouse embryonic stem (ES) cells (derived from C57Bl/6NTac mice) aretransfected by electroporation with the 3′ vector (805) according towidely used procedures. Prior to electroporation, the vector DNA islinearized with a rare-cutting restriction enzyme that cuts only in theprokaryotic plasmid sequence or the polylinker associated with it. Thetransfected cells are plated and after ˜24 hours they are placed underpositive selection for cells that have integrated the 3′ vector intotheir DNA by using the neomycin analogue drug G418. There is alsonegative selection for cells that have integrated the vector into theirDNA but not by homologous recombination. Non-homologous recombinationwill result in retention of the DTA gene (823), which will kill thecells when the gene is expressed, whereas the DTA gene is deleted byhomologous recombination since it lies outside of the region of vectorhomology with the mouse Igh locus. Colonies of drug-resistant ES cellsare physically extracted from their plates after they became visible tothe naked eye about a week later. These picked colonies aredisaggregated, re-plated in micro-well plates, and cultured for severaldays. Thereafter, each of the clones of cells is divided such that someof the cells can be frozen as an archive, and the rest used forisolation of DNA for analytical purposes.

DNA from the ES cell clones is screened by PCR using a widely practicedgene-targeting assay design. For this assay, one of the PCRoligonucleotide primer sequences maps outside the region of identityshared between the 3′ vector (805) and the genomic DNA, while the othermaps within the novel DNA between the two arms of genomic identity inthe vector, i.e., in the HPRT (845) or neomycin resistance (847) genes.According to the standard design, these assays detect pieces of DNA thatwould only be present in clones of ES cells derived from transfectedcells that undergo fully legitimate homologous recombination between the3′ targeting vector and the endogenous mouse Igh locus. Two separatetransfections are performed with the 3′ vector (805). PCR-positiveclones from the two transfections are selected for expansion followed byfurther analysis using Southern blot assays.

The Southern blot assays are performed according to widely usedprocedures using three probes and genomic DNA digested with multiplerestriction enzymes chosen so that the combination of probes and digestsallow the structure of the targeted locus in the clones to be identifiedas properly modified by homologous recombination. One of the probes mapsto DNA sequence flanking the 5′ side of the region of identity sharedbetween the 3′ targeting vector and the genomic DNA; a second probe mapsoutside the region of identity but on the 3′ side; and the third probemaps within the novel DNA between the two arms of genomic identity inthe vector, i.e., in the HPRT (845) or neomycin resistance (847) genes.The Southern blot identifies the presence of the expected restrictionenzyme-generated fragment of DNA corresponding to the correctly mutated,i.e., by homologous recombination with the 3′ Igh targeting vector, partof the Igh locus as detected by one of the external probes and by theneomycin or HPRT probe. The external probe detects the mutant fragmentand also a wild-type fragment from the non-mutant copy of theimmunoglobulin Igh locus on the homologous chromosome.

Karyotypes of PCR- and Southern blot-positive clones of ES cells areanalyzed using an in situ fluorescence hybridization procedure designedto distinguish the most commonly arising chromosomal aberrations thatarise in mouse ES cells. Clones with such aberrations are excluded fromfurther use. ES cell clones that are judged to have the expected correctgenomic structure based on the Southern blot data—and that also do nothave detectable chromosomal aberrations based on the karyotypeanalysis—are selected for further use.

Acceptable clones are then modified with the 5′ vector (803) usingprocedures and screening assays that are essentially identical in designto those used with the 3′ vector (805) except that puromycin selectionis used instead of G418/neomycin for selection. The PCR assays, probesand digests are also tailored to match the genomic region being modifiedby the 5′ vector (805).

Clones of ES cells that have been mutated in the expected fashion byboth the 3′ and the 5′ vectors, i.e., doubly targeted cells carryingboth engineered mutations, are isolated following vector targeting andanalysis. The clones must have undergone gene targeting on the samechromosome, as opposed to homologous chromosomes (i.e., the engineeredmutations created by the targeting vectors must be in cis on the sameDNA strand rather than in trans on separate homologous DNA strands).Clones with the cis arrangement are distinguished from those with thetrans arrangement by analytical procedures such as fluorescence in situhybridization of metaphase spreads using probes that hybridize to thenovel DNA present in the two gene targeting vectors (803 and 805)between their arms of genomic identity. The two types of clones can alsobe distinguished from one another by transfecting them with a vectorexpressing the Cre recombinase, which deletes the pu-TK (839), HPRT(845) and neomycin resistance (847) genes if the targeting vectors havebeen integrated in cis, and then comparing the number of colonies thatsurvive ganciclovir selection against the thymidine kinase geneintroduced by the 5′ vector (803) and by analyzing the drug resistancephenotype of the surviving clones by a “sibling selection” screeningprocedure in which some of the cells from the clone are tested forresistance to puromycin or G418/neomycin. Cells with the cis arrangementof mutations are expected to yield approximately 10³ moreganciclovir-resistant clones than cells with the trans arrangement. Themajority of the resulting cis-derived ganciclovir-resistant clones arealso sensitive to both puromycin and G418/neomycin, in contrast to thetrans-derived ganciclovir-resistant clones, which should retainresistance to both drugs. Doubly targeted clones of cells with thecis-arrangement of engineered mutations in the heavy chain locus areselected for further use.

The doubly targeted clones of cells are transiently transfected with avector expressing the Cre recombinase and the transfected cellssubsequently are placed under ganciclovir selection, as in theanalytical experiment summarized above. Ganciclovir-resistant clones ofcells are isolated and analyzed by PCR and Southern blot for thepresence of the expected deletion between the two engineered mutationscreated by the 5′ (803) and the 3′ (805) targeting vectors. In theseclones, the Cre recombinase causes a recombination (802) to occurbetween the loxP sites (837) introduced into the heavy chain locus bythe two vectors to create the genomic DNA configuration shown at 807.Because the loxP sites are arranged in the same relative orientations inthe two vectors, recombination results in excision of a circle of DNAcomprising the entire genomic interval between the two loxP sites. Thecircle does not contain an origin of replication and thus is notreplicated during mitosis and therefore is lost from the cells as theyundergo proliferation. The resulting clones carry a deletion of the DNAthat was originally between the two loxP sites. Clones that have theexpected deletion are selected for further use.

ES cell clones carrying the deletion of sequence in one of the twohomologous copies of their immunoglobulin heavy chain locus areretransfected (804) with a Cre recombinase expression vector togetherwith a piece of DNA (809) comprising a partly canine immunoglobulinheavy chain locus containing part-canine/part-mouse V_(H), D_(H) andJ_(H) region gene segments. The key features of this piece of syntheticDNA (809) are the following: a lox5171 site (831); a neomycin resistancegene open reading frame (847) lacking the initiator methionine codon,but in-frame and contiguous with an uninterrupted open reading frame inthe lox5171 site a FRT site (827); an array of 47 functional canineV_(H) heavy chain variable region genes (851), each comprised of caninecoding sequences embedded in mouse noncoding sequences; optionally a21.6 kb pre-D region from the mouse heavy chain locus (not shown); a 58Kb piece of DNA containing the 6 canine D_(H) gene segments (853) and 5canine J_(H) gene segments (855) where the canine coding sequences areembedded in mouse noncoding sequences; a loxP site (837) in oppositerelative orientation to the lox5171 site (831).

The transfected clones are placed under G418 selection, which enrichesfor clones of cells that have undergone RMCE in which the engineeredpartly canine donor immunoglobulin locus (809) is integrated in itsentirety into the deleted endogenous immunoglobulin heavy chain locusbetween the lox5171 (831) and loxP (837) sites to create the DNA regionillustrated at 811. Only cells that have properly undergone RMCE havethe capability to express the neomycin resistance gene (847) because thepromoter (829) as well as the initiator methionine codon (835) requiredfor its expression are not present in the vector (809) and are alreadypre-existing in the host cell Igh locus (807). The remaining elementsfrom the 5′ vector (803) are removed via Flp-mediated recombination(806) in vitro or in vivo, resulting in the final canine-based locus asshown at 813.

G418-resistant ES cell clones are analyzed by PCR and Southern blot todetermine if they have undergone the expected RMCE process withoutunwanted rearrangements or deletions. Clones that have the expectedgenomic structure are selected for further use.

ES cell clones carrying the partly canine immunoglobulin heavy chain DNA(813) in the mouse heavy chain locus are microinjected into mouseblastocysts from strain DBA/2 to create partially ES cell-derivedchimeric mice according to standard procedures. Male chimeric mice withthe highest levels of ES cell-derived contribution to their coats areselected for mating to female mice. The female mice of choice here areof C57Bl/6NTac strain, and also carry a transgene encoding the Flprecombinase that is expressed in their germline. Offspring from thesematings are analyzed for the presence of the partly canineimmunoglobulin heavy chain locus, and for loss of the FRT-flankedneomycin resistance gene that was created in the RMCE step. Mice thatcarry the partly canine locus are used to establish a colony of mice.

Example 4 Introduction of an Engineered Partly Canine ImmunoglobulinLocus into the Immunoglobulin Kappa Chain Gene Locus of a Mouse Genome

Another method for replacing a portion of a mouse genome with partlycanine immunoglobulin locus is illustrated in FIG. 9. This methodprovides introducing a first site-specific recombinase recognitionsequence into the mouse genome, which may be introduced either 5′ or 3′of the cluster of endogenous V_(K) (915) and J_(K) (919) region genesegments of the mouse genome, followed by the introduction of a secondsite-specific recombinase recognition sequence into the mouse genome,which in combination with the first sequence-specific recombination siteflanks the entire locus comprising clusters of V_(K) and J_(K) genesegments upstream of the constant region genes (921). The flanked regionis deleted and then replaced with a partly canine immunoglobulin locususing the relevant site-specific recombinase, as described herein.

The targeting vectors employed for introducing the site-specificrecombination sequences on either side of the V_(K) (915) and J_(K)(919) gene segments also include an additional site-specificrecombination sequence that has been modified so that it is stillrecognized efficiently by the recombinase, but does not recombine withunmodified sites. This site is positioned in the targeting vector suchthat after deletion of the V_(K) and J_(K) gene segment clusters it canbe used for a second site specific recombination event in which anon-native piece of DNA is moved into the modified V_(K) locus via RMCE.In this example, the non-native DNA is a synthetic nucleic acidcomprising both canine and mouse Igκ variable region gene sequences.

Two gene targeting vectors are constructed to accomplish the processjust outlined. One of the vectors (903) comprises mouse genomic DNAtaken from the 5′ end of the locus, upstream of the most distal V_(K)gene segment. The other vector (905) comprises mouse genomic DNA takenfrom within the locus downstream (3′) of the J_(K) gene segments (919)and upstream of the constant region genes (921).

The key features of the 5′ vector (903) are as follows: a gene encodingthe diphtheria toxin A (DTA) subunit under transcriptional control of amodified herpes simplex virus type I thymidine kinase gene promotercoupled to two mutant transcriptional enhancers from the polyoma virus(923); 6 Kb of mouse genomic DNA (925) mapping upstream of the mostdistal variable region gene in the kappa chain locus; a FRT recognitionsequence for the Flp recombinase (927); a piece of genomic DNAcontaining the mouse Polr2a gene promoter (929); a translationinitiation sequence (935, methionine codon embedded in a “Kozak”consensus sequence); a mutated loxP recognition sequence (lox5171) forthe Cre recombinase (931); a transcription termination/polyadenylationsequence (933); a loxP recognition sequence for the Cre recombinase(937); a gene encoding a fusion protein comprised of a proteinconferring resistance to puromycin fused to a truncated form of thethymidine kinase (pu-TK) under transcriptional control of the promoterfrom the mouse phosphoglycerate kinase 1 gene (939); 2.5 Kb of mousegenomic DNA (941) mapping close to the 6 Kb sequence at the 5′ end inthe vector and arranged in the native relative orientation.

The key features of the 3′ vector (905) are as follows: 6 Kb of mousegenomic DNA (943) mapping within the intron between the J_(κ) (919) andC_(κ) (921) gene loci; a gene encoding the human hypoxanthine-guaninephosphoribosyl transferase (HPRT) under transcriptional control of themouse Polr2a gene promoter (945); a neomycin resistance gene under thecontrol of the mouse phosphoglycerate kinase 1 gene promoter (947); aloxP recognition sequence for the Cre recombinase (937); 3.6 Kb of mousegenomic DNA (949) that maps immediately downstream in the genome of the6 Kb DNA fragment included at the 5′ end in the vector, with the twofragments oriented in the same relative way as in the mouse genome; agene encoding the diphtheria toxin A (DTA) subunit under transcriptionalcontrol of a modified herpes simplex virus type I thymidine kinase genepromoter coupled to two mutant transcriptional enhancers from thepolyoma virus (923).

Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice aretransfected by electroporation with the 3′ vector (905) according towidely used procedures. Prior to electroporation, the vector DNA islinearized with a rare-cutting restriction enzyme that cuts only in theprokaryotic plasmid sequence or the polylinker associated with it. Thetransfected cells are plated and after ˜24 hours they are placed underpositive selection for cells that have integrated the 3′ vector intotheir DNA by using the neomycin analogue drug G418. There is alsonegative selection for cells that have integrated the vector into theirDNA but not by homologous recombination. Non-homologous recombinationwill result in retention of the DTA gene, which will kill the cells whenthe gene is expressed, whereas the DTA gene is deleted by homologousrecombination since it lies outside of the region of vector homologywith the mouse Igκ locus. Colonies of drug-resistant ES cells arephysically extracted from their plates after they became visible to thenaked eye about a week later. These picked colonies are disaggregated,re-plated in micro-well plates, and cultured for several days.Thereafter, each of the clones of cells is divided such that some of thecells could be frozen as an archive, and the rest used for isolation ofDNA for analytical purposes.

DNA from the ES cell clones is screened by PCR using a widely usedgene-targeting assay design. For this assay, one of the PCRoligonucleotide primer sequences maps outside the region of identityshared between the 3′ vector (905) and the genomic DNA (901), while theother maps within the novel DNA between the two arms of genomic identityin the vector, i.e., in the HPRT (945) or neomycin resistance (947)genes. According to the standard design, these assays detect pieces ofDNA that are only present in clones of ES cells derived from transfectedcells that had undergone fully legitimate homologous recombinationbetween the 3′ vector (905) and the endogenous mouse Igic locus. Twoseparate transfections are performed with the 3′ vector (905).PCR-positive clones from the two transfections are selected forexpansion followed by further analysis using Southern blot assays.

The Southern blot assays are performed according to widely usedprocedures; they involve three probes and genomic DNA digested withmultiple restriction enzymes chosen so that the combination of probesand digests allowed for conclusions to be drawn about the structure ofthe targeted locus in the clones and whether it is properly modified byhomologous recombination. One of the probes maps to DNA sequenceflanking the 5′ side of the region of identity shared between the 3′kappa targeting vector (905) and the genomic DNA; a second probe alsomaps outside the region of identity but on the 3′ side; the third probemaps within the novel DNA between the two arms of genomic identity inthe vector, i.e., in the HPRT (945) or neomycin resistance (947) genes.The Southern blot identifies the presence of the expected restrictionenzyme-generated fragment of DNA corresponding to the correctly mutated,i.e., by homologous recombination with the 3′ kappa targeting vector(905) part of the kappa locus, as detected by one of the external probesand by the neomycin resistance or HPRT gene probe. The external probedetects the mutant fragment and also a wild-type fragment from thenon-mutant copy of the immunoglobulin kappa locus on the homologouschromosome.

Karyotypes of PCR- and Southern blot-positive clones of ES cells areanalyzed using an in situ fluorescence hybridization procedure designedto distinguish the most commonly arising chromosomal aberrations thatarise in mouse ES cells. Clones with such aberrations are excluded fromfurther use. Karyoptypically normal clones that are judged to have theexpected correct genomic structure based on the Southern blot data areselected for further use.

Acceptable clones are then modified with the 5′ vector (903) usingprocedures and screening assays that are essentially identical in designto those used with the 3′ vector (905), except that puromycin selectionis used instead of G418/neomycin selection, and the protocols aretailored to match the genomic region modified by the 5′ vector (903).The goal of the 5′ vector (903) transfection experiments is to isolateclones of ES cells that have been mutated in the expected fashion byboth the 3′ vector (905) and the 5′ vector (903), i.e., doubly targetedcells carrying both engineered mutations. In these clones, the Crerecombinase causes a recombination (902) to occur between the loxP sitesintroduced into the kappa locus by the two vectors, resulting in thegenomic DNA configuration shown at 907.

Further, the clones must have undergone gene targeting on the samechromosome, as opposed to homologous chromosomes; i.e., the engineeredmutations created by the targeting vectors must be in cis on the sameDNA strand rather than in trans on separate homologous DNA strands.Clones with the cis arrangement are distinguished from those with thetrans arrangement by analytical procedures such as fluorescence in situhybridization of metaphase spreads using probes that hybridize to thenovel DNA present in the two gene targeting vectors (903 and 905)between their arms of genomic identity. The two types of clones can alsobe distinguished from one another by transfecting them with a vectorexpressing the Cre recombinase, which deletes the pu-Tk (939), HPRT(945) and neomycin resistance (947) genes if the targeting vectors havebeen integrated in cis, and comparing the number of colonies thatsurvive ganciclovir selection against the thymidine kinase geneintroduced by the 5′ vector (903) and by analyzing the drug resistancephenotype of the surviving clones by a “sibling selection” screeningprocedure in which some of the cells from the clone are tested forresistance to puromycin or G418/neomycin. Cells with the cis arrangementof mutations are expected to yield approximately 10³ moreganciclovir-resistant clones than cells with the trans arrangement. Themajority of the resulting cis-derived ganciclovir-resistant clonesshould also be sensitive to both puromycin and G418/neomycin, incontrast to the trans-derived ganciclovir-resistant clones, which shouldretain resistance to both drugs. Clones of cells with thecis-arrangement of engineered mutations in the kappa chain locus areselected for further use.

The doubly targeted clones of cells are transiently transfected with avector expressing the Cre recombinase (902) and the transfected cellsare subsequently placed under ganciclovir selection, as in theanalytical experiment summarized above. Ganciclovir-resistant clones ofcells are isolated and analyzed by PCR and Southern blot for thepresence of the expected deletion (907) between the two engineeredmutations created by the 5′ vector (903) and the 3′ vector (905). Inthese clones, the Cre recombinase has caused a recombination to occurbetween the loxP sites (937) introduced into the kappa chain locus bythe two vectors. Because the loxP sites are arranged in the samerelative orientations in the two vectors, recombination results inexcision of a circle of DNA comprising the entire genomic intervalbetween the two loxP sites. The circle does not contain an origin ofreplication and thus is not replicated during mitosis and is thereforelost from the clones of cells as they undergo clonal expansion. Theresulting clones carry a deletion of the DNA that was originally betweenthe two loxP sites. Clones that have the expected deletion are selectedfor further use.

The ES cell clones carrying the deletion of sequence in one of the twohomologous copies of their immunoglobulin kappa chain locus areretransfected (904) with a Cre recombinase expression vector togetherwith a piece of DNA (909) comprising a partly canine immunoglobulinkappa chain locus containing Vκ (951) and Jκ (955) gene segments. Thekey features of this piece of DNA (referred to as “K-K”) are thefollowing: a lox5171 site (931); a neomycin resistance gene open readingframe (947, lacking the initiator methionine codon, but in-frame andcontiguous with an uninterrupted open reading frame in the lox5171 site(931)); a FRT site (927); an array of 19 canine Vκ gene segments (951),each comprised of canine coding sequences embedded in mouse noncodingsequences; optionally a 13.5 Kb piece of genomic DNA from immediatelyupstream of the cluster of J kappa region gene segments in the mousekappa chain locus (not shown); a 2 Kb piece of DNA containing the 5canine Jκ region gene segments (955) embedded in mouse noncoding DNA; aloxP site (937) in opposite relative orientation to the lox5171 site(931).

The sequences of the canine Vκ and Jκ gene coding regions are in Table2.

In a second independent experiment, an alternative piece of partlycanine DNA (909) is used in place of the K-K DNA. The key features ofthis DNA (referred to as “L-K”) are the following: a lox5171 site (931);a neomycin resistance gene open reading frame (947) lacking theinitiator methionine codon, but in-frame and contiguous with anuninterrupted open reading frame in the lox5171 site (931); a FRT site(927); an array of 7 functional canine V_(λ) variable region genesegments (951), each comprised of canine coding sequences embedded inmouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kbpiece of genomic DNA from immediately upstream of the cluster of the Jregion gene segments in the mouse kappa chain locus (not shown); a 2 Kbpiece of DNA containing five canine Jλ region gene segments embedded inmouse noncoding DNA (955); a loxP site (937) in opposite relativeorientation to the lox5171 site (931).

The transfected clones from the K-K and L-K transfection experiments areplaced under G418 selection, which enriches for clones of cells thathave undergone RMCE, in which the partly canine donor DNA (909) isintegrated in its entirety into the deleted immunoglobulin kappa chainlocus between the lox5171 (931) and loxP (937) sites that were placedthere by 5′ (903) and 3′ (905) vectors, respectively. Only cells thathave properly undergone RMCE have the capability to express the neomycinresistance gene (947) because the promoter (929) as well as theinitiator methionine codon (935) required for its expression are notpresent in the vector (909) and are already pre-existing in the hostcell Igh locus (907). The DNA region created using the K-K sequence isillustrated at 911. The remaining elements from the 5′ vector (903) areremoved via Flp-mediated recombination (906) in vitro or in vivo,resulting in the final canine-based light chain locus as shown at 913.

G418-resistant ES cell clones are analyzed by PCR and Southern blottingto determine if they have undergone the expected RMCE process withoutunwanted rearrangements or deletions. Both K-K and L-K clones that havethe expected genomic structure are selected for further use.

The K-K ES cell clones and the L-K ES cell clones carrying the partlycanine immunoglobulin DNA in the mouse kappa chain locus (913) aremicroinjected into mouse blastocysts from strain DBA/2 to create partlyES cell-derived chimeric mice according to standard procedures. Malechimeric mice with the highest levels of ES cell-derived contribution totheir coats are selected for mating to female mice. The female mice ofchoice for use in the mating are of the C57Bl/6NTac strain, and alsocarry a transgene encoding the Flp recombinase that is expressed intheir germline. Offspring from these matings are analyzed for thepresence of the partly canine immunoglobulin kappa or lambda light chainlocus, and for loss of the FRT-flanked neomycin resistance gene that wascreated in the RMCE step. Mice that carry the partly canine locus areused to establish colonies of K-K and L-K mice.

Mice carrying the partly canine heavy chain locus, produced as describedin Example 3, can be bred with mice carrying a canine-based kappa chainlocus. Their offspring are in turn bred together in a scheme thatultimately produces mice that are homozygous for both canine-based loci,i.e., canine-based for heavy chain and kappa. Such mice produce partlycanine heavy chains comprised of canine variable domains and mouseconstant domains. They also produce partly canine kappa proteinscomprised of canine kappa variable domains and the mouse kappa constantdomain from their kappa loci. Monoclonal antibodies recovered from thesemice are comprised of canine heavy chain variable domains paired withcanine kappa variable domains.

A variation on the breeding scheme involves generating mice that arehomozygous for the canine-based heavy chain locus, but heterozygous atthe kappa locus such that on one chromosome they have the K-Kcanine-based locus and on the other chromosome they have the L-Kcanine-based locus. Such mice produce partly canine heavy chainscomprised of canine variable domains and mouse constant domains. Theyalso produce partly canine kappa proteins comprised of canine kappavariable domains and the mouse kappa constant domain from one of theirkappa loci. From the other kappa locus, they will produce partly caninelambda proteins comprised of canine lambda variable domains the mousekappa constant domain. Monoclonal antibodies recovered from these miceare comprised of canine variable domains paired in some cases withcanine kappa variable domains and in other cases with canine lambdavariable domains.

Example 5 Introduction of an Engineered Partly Canine ImmunoglobulinLocus into the Immunoglobulin Lambda Chain Gene Locus of a Mouse Genome

Another method for replacing a portion of a mouse genome with anengineered partly canine immunoglobulin locus is illustrated in FIG. 10.This method comprises deleting approximately 194 Kb of DNA from thewild-type mouse immunoglobulin lambda locus (1001)—comprising Vλx/Vλ2gene segments (1013), Jλ2/Cλ2 gene cluster (1015), and Vλ1 gene segment(1017)—by a homologous recombination process involving a targetingvector (1003) that shares identity with the locus both upstream of theVλx/Vλ2 gene segments (1013) and downstream of the Vλ1 gene segment(1017) in the immediate vicinity of the J3, C3, J1 and C1 lambda genecluster (1023). The vector replaces the 194 Kb of DNA with elementsdesigned to permit a subsequent site-specific recombination in which anon-native piece of DNA is moved into the modified Vλ locus via RMCE(1004). In this example, the non-native DNA is a synthetic nucleic acidcomprising both canine and mouse sequences.

The key features of the gene targeting vector (1003) for accomplishingthe 194 Kb deletion are as follows: a negative selection gene such as agene encoding the A subunit of the diphtheria toxin (DTA, 1059) or aherpes simplex virus thymidine kinase gene (not shown); 4 Kb of genomicDNA from 5′ of the mouse Vλx/Vλ2 variable region gene segments in thelambda locus (1025); a FRT site (1027); a piece of genomic DNAcontaining the mouse Polr2a gene promoter (1029); a translationinitiation sequence (methionine codon embedded in a “Kozak” consensussequence) (1035); a mutated loxP recognition sequence (lox5171) for theCre recombinase; a transcription termination/polyadenylation sequence(1033); an open reading frame encoding a protein that confers resistanceto puromycin (1037), whereas this open reading frame is on the antisensestrand relative to the Polr2a promoter and the translation initiationsequence next to it and is followed by its own transcriptiontermination/polyadenylation sequence (1033); a loxP recognition sequencefor the Cre recombinase (1039); a translation initiation sequence (amethionine codon embedded in a “Kozak” consensus sequence) (1035) on thesame, antisense strand as the puromycin resistance gene open readingframe; a chicken beta actin promoter and cytomegalovirus early enhancerelement (1041) oriented such that it directs transcription of thepuromycin resistance open reading frame, with translation initiating atthe initiation codon downstream of the loxP site and continuing backthrough the loxP site into the puromycin open reading frame all on theantisense strand relative to the Polr2a promoter and the translationinitiation sequence next to it; a mutated recognition site for the Flprecombinase known as an “F3” site (1043); a piece of genomic DNAupstream of the J3, C3, J1 and C1 lambda gene segments (1045).

Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice aretransfected (1002) by electroporation with the targeting vector (1003)according to widely used procedures. Homologous recombination replacesthe native DNA with the sequences from the targeting vector (1003) inthe 196 Kb region resulting in the genomic DNA configuration depicted at1005.

Prior to electroporation, the vector DNA is linearized with arare-cutting restriction enzyme that cuts only in the prokaryoticplasmid sequence or the polylinker associated with it. The transfectedcells are plated and after ˜24 hours placed under positive drugselection using puromycin. There is also negative selection for cellsthat have integrated the vector into their DNA but not by homologousrecombination. Non-homologous recombination will result in retention ofthe DTA genes, which will kill the cells when the genes are expressed,whereas the DTA genes are deleted by homologous recombination since theylie outside of the region of vector homology with the mouse Igl locus.Colonies of drug-resistant ES cells are physically extracted from theirplates after they became visible to the naked eye over a week later.These picked colonies are disaggregated, re-plated in micro-well plates,and cultured for several days. Thereafter, each of the clones of cellsare divided such that some of the cells are frozen as an archive, andthe rest used for isolation of DNA for analytical purposes.

DNA from the ES cell clones is screened by PCR using a widely usedgene-targeting assay design. For these assays, one of the PCRoligonucleotide primer sequences maps outside the regions of identityshared between the targeting vector and the genomic DNA, while the othermaps within the novel DNA between the two arms of genomic identity inthe vector, e.g., in the puro gene (1037). According to the standarddesign, these assays detect pieces of DNA that would only be present inclones of cells derived from transfected cells that had undergone fullylegitimate homologous recombination between the targeting vector (1003)and the native DNA (1001).

Six PCR-positive clones from the transfection (1002) are selected forexpansion followed by further analysis using Southern blot assays. TheSouthern blots involve three probes and genomic DNA from the clones thathas been digested with multiple restriction enzymes chosen so that thecombination of probes and digests allow identification of whether the EScell DNA has been properly modified by homologous recombination.

Karyotypes of the six PCR- and Southern blot-positive clones of ES cellsare analyzed using an in situ fluorescence hybridization proceduredesigned to distinguish the most commonly arising chromosomalaberrations that arise in mouse ES cells. Clones that show evidence ofaberrations are excluded from further use. Karyoptypically normal clonesthat are judged to have the expected correct genomic structure based onthe Southern blot data are selected for further use.

The ES cell clones carrying the deletion in one of the two homologouscopies of their immunoglobulin lambda chain locus are retransfected(1004) with a Cre recombinase expression vector together with a piece ofDNA (1007) comprising a partly canine immunoglobulin lambda chain locuscontaining V, J and C region gene segments. The key features of thispiece of DNA (1007) are as follows: a lox5171 site (1031); a neomycinresistance gene open reading frame lacking the initiator methioninecodon, but in-frame and contiguous with an uninterrupted open readingframe in the lox5171 site (1047); a FRT site 1027); an array of 7functional canine lambda variable region gene segments, each comprisedof canine lambda coding sequences embedded in mouse lambda noncodingsequences (1051); an array of J-C units where each unit is comprised ofa canine Jλ gene segment and a mouse lambda constant domain gene segmentembedded within noncoding sequences from the mouse lambda locus (1055)(the canine Jλ gene segments are those encoding Jλ1, Jλ2, Jλ6 and Jλ7,while the mouse lambda constant domain gene segments are C1 and/or C2and/or C3); a mutated recognition site for the Flp recombinase known asan “F3” site (1043); an open reading frame conferring hygromycinresistance (1057), which is located on the antisense strand relative tothe immunoglobulin gene segment coding information in the construct; aloxP site (1039) in opposite relative orientation to the lox5171 site.

The sequences of the canine Vλ and Jλ gene coding regions are in Table3.

The transfected clones are placed under G418 and/or hygromycinselection, which enriches for clones of cells that have undergone a RMCEprocess, in which the partly canine donor DNA is integrated in itsentirety into the deleted immunoglobulin lambda chain locus between thelox5171 and loxP sites that were placed there by the gene targetingvector. The remaining elements from the targeting vector (1003) areremoved via FLP-mediated recombination (1006) in vitro or in vivoresulting in the final caninized locus as shown at 1011.

G418/hygromycin-resistant ES cell clones are analyzed by PCR andSouthern blotting to determine if they have undergone the expectedrecombinase-mediated cassette exchange process without unwantedrearrangements or deletions. Clones that have the expected genomicstructure are selected for further use.

The ES cell clones carrying the partly canine immunoglobulin DNA (1011)in the mouse lambda chain locus are microinjected into mouse blastocystsfrom strain DBA/2 to create partially ES cell-derived chimeric miceaccording to standard procedures. Male chimeric mice with the highestlevels of ES cell-derived contribution to their coats are selected formating to female mice. The female mice of choice here are of theC57Bl/6NTac strain, which carry a transgene encoding the Flp recombinaseexpressed in their germline. Offspring from these matings are analyzedfor the presence of the partly canine immunoglobulin lambda chain locus,and for loss of the FRT-flanked neomycin resistance gene and theF3-flanked hygromycin resistance gene that were created in the RMCEstep. Mice that carry the partly canine locus are used to establish acolony of mice.

In some aspects, the mice comprising the canine-based heavy chain andkappa locus (as described in Examples 3 and 4) are bred to mice thatcarry the canine-based lambda locus. Mice generated from this type ofbreeding scheme are homozygous for the canine-based heavy chain locus,and can be homozygous for the K-K canine-based locus or the L-Kcanine-based locus. Alternatively, they can be heterozygous at the kappalocus carrying the K-K locus on one chromosome and the L-K locus on theother chromosome. Each of these mouse strains is homozygous for thecanine-based lambda locus. Monoclonal antibodies recovered from thesemice are comprised of canine heavy chain variable domains paired in somecases with canine kappa variable domains and in other cases with caninelambda variable domains. The lambda variable domains are derived fromeither the canine-based L-K locus or the canine-based lambda locus.

Example 6 Introduction of an Engineered Partly Canine ImmunoglobulinMinilocus into a Mouse Genome

In certain other aspects, the partly canine immunoglobulin locuscomprises a canine variable domain minilocus such as the one illustratedin FIG. 11. Here instead of a partly canine immunoglobulin locuscomprising all or substantially all of the canine V_(H) gene segmentcoding sequences, the mouse immunoglobulin locus is replaced with aminilocus (1119) comprising fewer chimeric canine V_(H) gene segments,e.g. 1-79 canine V_(H) gene segments determined to be functional; thatis, not pseudogenes.

A site-specific targeting vector (1131) comprising the partly canineimmunoglobulin locus to be integrated into the mammalian host genome isintroduced (1102) into the genomic region (1101) with the deletedendogenous immunoglobulin locus comprising the puro-TK gene (1105) andthe following flanking sequence-specific recombination sites: mutant FRTsite (1109), mutant LoxP site (1111), wild-type FRT site (1107), andwild-type LoxP site (1105). The site-specific targeting vector comprisesi) an array of optional PAIR elements (1141); ii) a V_(H) locus (1119)comprising, e.g., 1-47 functional canine V_(H) coding regions andintervening sequences based on the mouse genome endogenous sequences;iii) a 21.6 kb pre-D region (1121) comprising mouse sequence; iv) aD_(H) locus (1123) and a J_(H) locus (1125) comprising 6 D_(H) and 5J_(H) canine coding sequences and intervening sequences based on themouse genome endogenous sequences. The partly canine immunoglobulinlocus is flanked by recombination sites—mutant FRT (1109), mutant LoxP(1111), wild-type FRT (1107), and wild-type LoxP (1105)—that allowrecombination with the modified endogenous locus. Upon introduction ofthe appropriate recombinase, e.g., Cre) (1104), the partly canineimmunoglobulin locus is integrated into the genome upstream of theconstant gene region (1127) as shown at 1129.

As described in Example 1, the primary screening for introduction of thepartly canine immunoglobulin variable region locus is carried out byprimary PCR screens supported by secondary Southern blotting assays. Thedeletion of the puro-TK gene (1105) as part of the recombination eventallows identification of the cells that did not undergo therecombination event using ganciclovir negative selection.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims. In the claims thatfollow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112 ¶6. All references cited hereinare incorporated by reference in their entirety for all purposes.

TABLE 1 Canine IgH locus Germline VII sequences(NB, the sequence and annotation of the dog genome is still incomplete.This table does not necessarily describe the complete canine VH, DHand JH gene repertoire.) SEQ ID NO. 1 vhlccttgcacag taatacactg ccgtgtcctc atctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagct 120tgtgctactt ccatcactgc taatttgtga gacccactgc agccccttcc ctggagcctg 180gcggatccag ctcatgtagt tgctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 2 vh2ctttgcacag taatacaccg ctgtgtcctc agctctcagg ctgttcatct gcagatacag 60cgtattcttt gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120tgtgctactt ccatctttgt taatgtgtga gacccactga agccccttcc ctggagcctg 180gcgggcccaa tacatgtagt aactactgaa ggtaaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttca ccatgtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 3 vh3ggagtgaaca cagacaaacc accatgaagt tgttgctctg ctgggctttt cttcttgcaa 60tttaaaaagt aattcatgga gaacaagaga ccttgaatat atgagttgag ttaagtgaga 120gaaacagggg atgtgggaca gtttcctgac caggatgtct tgtgtctgca ggtgtccagg 180gtgaggtgca gctggtggag tctgggggag acctgatgaa gcctgggggg gtccctgaga 240ctctcctgtg tggcctctga attcatcttc agtggctact ggaagtactg gatccaccaa 300gctccaggga aggggctgca gtgggtcaca tggattagca atgatggaag tagcaaaagc 360tatgcagacg ctgtgaaggg ccaattcacc atctccaaag acaatgccaa atacacgctg 420tatctgcaga tgaacagcct gagagccgag gacatggccg tgtattactg tatga 475 //SEQ ID NO. 4 vh4 pseudoaggtgcagct ggtggagtct gggggagacc tgatgaagcc tgggggggtc cctgagactc 60tcctgtgtgg cctctgaatt catcttcagt ggctactgga agtactggat ccaccaagct 120ccagggaagg ggctgcagtg ggtcacatgg attagcaatg atggaagtag caaaagctat 180gcagacgctg tgaagggcca attcaccatc tccaaagaca atgccaaata cacgctgtat 240ctgcagatga acagcctgag agccgaggac atggccgtgt attactgtat ga 292 //SEQ ID NO. 5 vh5 pseudoaatctgaggt ccagctggtg cagtctgggg ctgaggtgag gaaaccagtt tcatctgtga 60aggtctcctg gaaggcatct ggatacacct acatggatgc ttatatgcac tggttatgac 120aagcttcagg aataaggttt gggtgtatgg gatggattgg tcccaaagat ggtgccacaa 180gatattcaca gaagttccac agcagagtct ccctgatggc agacatgtcc aaagcacagc 240ctacatgctg ctgagcagtc agaggcctga ggacacacct gcatattact gtgt 294 //SEQ ID NO. 6 vh6actcgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gaagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120tgtgctactt ccaccactgt taatgtatgc gacccactga agccccttcc ctggagcctg 180gcggacccag ctcatgtggt agctactgaa ggtgaatcca gaggccacac aggaaagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 7 vh7gtctgcacag taatacacgg ccgtgtcctc ggctctgagg ctgttcatct gcagatagag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagct 120tgtgctactt ccatcattgc taatgtatgc gacccactga agcccctttc ctggagcctg 180gcggatccag ctcatgtcgg agctactgaa ggtgaatcca gaggctacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 8 vh8cctcatgcag taatacacgg ctgtgtcctc ggctctcagg ctgttcatct gcagatacag 60catgttcttg gtgttgtctc tggagatggt gaatctgtcc ctcaaagtgt ctgcgtagta 120tgtactactt ccatcatagc taatatgtcc gacccactgc agccccttct tttgagcctg 180gcggacccag ctcatgccat agctactgaa ggtgaatcca gaggcctgac aggagagacc 240cagggaaccc ccaggattca ccatgtgtcc tccaaactcc accagttgct cctc 294 //SEQ ID NO. 9 vh9actcgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacgc 60catgttcttg gcgttgtctc tggagatggt gaatcggccc tttacagctt ctgcatagta 120tgtggtactt ccactctcat aaatccttgc gacccactcc agccctttcc ctggagcctg 180gcggacccag tacatttcgt agttactgaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 10 vh10ccttgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagta 120tgtgctactt ccactactgc taatttctga gagccactgc agccccttcc ctggagcctg 180gcggacccag tccatgtcat agctactgaa ggtgaatcca gaggccacac aggaaagtct 240cagggacccc ccaggcttca ccaggtctcc tccagactcc accagctgca cctc 294 //SEQ ID NO. 11 vh11actcacacag taatacacgg ccatgtcctt gtctctcagg ctgttcatct gcagatagag 60cgtgttcctg gcgttgtctc tggagatggt gaattggccc ttcacagcgt ctgcatacct 120tgtgctactt ccaccattgc taatgtatgt gacccactgt aaccctttcc ctggagcctg 180gtagacccag tccatgtcgt agctactgaa ggtgaatcca gaggccacac aggaaagtct 240cagggatccc ccaggcttca ccaggtctcc ctcagtctcc accagctgca cctc 294 //SEQ ID NO. 12 vh12 pseudotttcacacaa taatacacag ccgtgtcctc ggctcccagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120tgtgctactt ctatcattgg taatacctgc gacccactgc agccccttcc ctggagccta 180gtggacccag cccatgtagt agctactgaa ggtgaatcta gaggccacac aggagaggga 240cctccccagg cttcaccacg tctcccctag actccaccag ctgcacctca ccct 294 //SEQ ID NO. 13 vh13 pseudocttcccacag taatacacag ctgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagta 120tgtgctactt ccaccactgt taatgtatcc gacccactgc agccccttcc ctggagcctg 180gcggacccag ctcatgtagt agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagagacccc ccaggcttca ccaggtctcc ccagactcca ccagctgcac ctca 294 //SEQ ID NO. 14 vh14cctcatgcag taatacacgg ctgtgtcctc ggctctcagg ctgttcatct gcagatacag 60catgttcttg gtgttgtctc tggagatggt gaatctgtcc ctcacagtgt ctgtgtagta 120tgtgctactt ccatcatagc taatatgtcc gacccactgc agccccttct tttgagcctg 180gcggacccag ctcatgccat agctactgaa ggtgaatcca gaggcctgac aggagagacc 240cagggaaccc ccaggattca ccatgtgtcc tccaaactcc accagttgct cctc 294 //SEQ ID NO. 15 vh15 pseudoactcacacag taatacacgg ccgtgtccta ggctctcagg ctgttcatct gcagatacac 60catgttcttg gcgttgtctc tggagatggt gaatcggccc tttacagctt ctgcgtagta 120tgtggtactt ccactctcat aaatccttgc gacccactcc agccctttcc ctggagcctg 180gtggacccag tacattttgt agttactgaa ggtgaatcca gaggccacac aggagagtct 240cagggatccc ccaggcttca ccaggtctcc cccagactcc accatctgca cctc 294 //SEQ ID NO. 16 vh16tctcgcacag taataaaggg ctgtgtcctc cactgtcagg ctgttcatct gcagatacag 60tgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgggtagta 120tgtgctactt ccatctttgt taatatatcc gacccactga ttgcccttcc ctggtgcctg 180gcggatccaa aacatgtagt aactactaaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc tccagactcc accagctgta cctc 294 //SEQ ID NO. 17 vh17cttcgcacag taatacacgg ctgtgtcctc agttctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgtgtaact 120tgtgctcctt ccatcactgc taatgtatgc gacccactgc agccccttcc ctggagcctg 180gcggacccag ctcatgtcgt agctactgaa ggtgaagcca gagaccacac aggagagtct 240cagggacccc tcaggtttca caaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 18 vh18ctttgcacag taatacatgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacac 60tgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgtgtagta 120tgtgctactt ccaccgctgt taatacctgc gaccaactgc agccccttct caggagcctg 180gcggacccag ctcatgctgt agctactgaa ggtgaatcca gaggccacac aggacagtct 240cagggacccc gcaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 19 vh19 pseudocacctgcaca gtaatacacc gctgtgtcct cactctcagg ctgttcatct gcagatacag 60tgtgttcttg gcattgtctc tggaggtggt gaatcagccc ttcacagcgt ctgtgtacct 120agtgctactt ccaccactgt tactgtatgc gactcactgc agcccctttc ctggagcctg 180gcagacccat cacatgcagt agatactgaa ggtgaatcca gaggacacag aggagagtct 240caggaccctc caggcttcac caggtatccc ccagactcca ccagctgcac ctca 294 //SEQ ID NO. 20 vh20 pseudoactctcacag taatacacgg ccgtgtcctc agctctcaag ctgttcatct gcaggtacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagtgt ctgtgtagta 120tgtgctcctt ccatcttagc taatagctgc gaccaactgt agccccttcc ccaaagcctg 180gcagacccag ctcatgccat acttactgaa ggtgaatcca gagaccacac aggacagtct 240gcagacccag ctcatgccat acttactgaa ggtgaatcca gagaccacac aggacagtct 294 //SEQ ID NO. 21 vh21 pseudocctcaatgtg tcactcacat aataatacag ggctctcagg ctgttcatat gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcacccc ttcacagcat ctgcatagct 120ttttctgctt ccaccagtat taacccatat gacccactgc agccccttcc ctggagcctg 180gctgacccag ctcatccagt agctcctgaa ggtgaatcca gaggtcacat aggagagtct 240aattgatccc ccaggcttca ccaggtctcc cccagactcc actagcttca cctc 294 //SEQ ID NO. 22 vh22 pseudogtctgcacag taatacatgg ccatgtcctc ggctctcagg ctgttcatct gcagatagag 60cgtgttcttg gcgttgtctc tggagatggt gaatcagccc ttcagagcgt ctacgtagct 120tgtgctactt ccatctgtgc taatacctgc gacccactgc agccccttcc ccggagtttg 180gtggatccag tacatccagt agctactgaa ggtgaatcca gaggccacac aggaggtctc 240agggatcctg caggcttcat cagttctccc ccagactcca tcatctgcac ctca 294 //SEQ ID NO. 23 vh23cttcgcacag gaatacacgg ctgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatacct 120tgtgctactt ccatcatacc taatcaatga gaccccctgc agccccttcc ctggagcctg 180gcggacccag ctcatgccgt agctactgaa ggtgaatcca gaggccacac aggacagtct 240cagggatccc ccaggcttct ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 24 vh24ccttgcacaa taatatacgg ccatgtcctc ggctctcagg ctgttcatct gcagatacag 60catgttcttg gcattgtctc tggagatggt gaatcggccc ttcacaatgt ctgcgtagct 120tgtgctactt ccactactgc taatttctgc aacccactgt agccccttcc ttggagcctg 180gcagaaccag ctcatgtaga agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc tcaggcttca caaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 25 vh25cttcacacag taatacacgg ccgtgtcctc ggatctcagg ctgttcatct gcagatacag 60cgtgttcttg acattgtctc tggagatggt gaattcaccc ttcacagcat ctgtgtagct 120tgtgctactt ccaccgctgt taaaacctgc gacccactgc atccccttcc ctggaggctg 180gtgagcccag cccatgttgt agctactgaa ggtgaatcca gggaccacac aggagagtct 240cagggacgcc ccaggcttca ccagttctcc cccaggctcc accagctgca cctc 294 //SEQ ID NO. 26 vh26ccttgcacag taatacatgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatgat gaatcggccc ttcacagcgt ctgcatagtt 120tgtgctactt ccactaccgc taatttctgc aacccactgt agccccttcc ctggagcctg 180gcggacccag ctcatccagt agctactgaa ggtgaatcca gagcccacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctaca cctc 294 //SEQ ID NO. 27 vh27 pseudogcacagtaat acacggccgt gtcctccctc ggctctcagg ctgttcttct acagatacag 60tgtgtttttg gcattgtctc tggagatggt gaatcggccc ttcagagcgt ctgcgtagct 120tgtgctactt ccatcatatc taatacctgc gaccccctgt agccccatcc cgggagcctc 180acgggcccac cacatgctgt agctactgaa ggtgaatcca gagcccacac aggagagtct 240cagggacctc ctaggcttca ccacgtctcc cgcagactcc accagatgca cctt 294 //SEQ ID NO. 28 vh28 pseudocctcacacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcgttgtctc tagaaacagt gaatcggcct ttcacagctt ctgcgtagct 120tgtgctactt ccatcatacc taatccatgc gacccatgga gccccttccc tggagcctgg 180tggacccagt acatccagta gctactgaag gtgaatccag agcccacaca ggagactctc 240agggaccccc ccagacttca ccaggtctcc cccagactcc actagctgca cctc 294 //SEQ ID NO. 29 vh29 pseudocctcacacag taatacaggg ccgtgtcatc ggctcccagg ctgctcatct gtagatacag 60cgtgttcttg gtgttgtctc tggagatggt aaatcggccc tttactgtgt ctgcgtgatt 120tgtgctactt ccactattgc taatagttgt gatccactgc agccccttcc ttggagcctg 180gcggacccag atcatgctgt agttactgaa ggtgaatccg gaagccacac aggagacagg 240agagtctcag gaaacctcca gtcttcacca ggtctcccca ggactccacc agct 294 //SEQ ID NO. 30 vh30cttcgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcaaatacag 60cgagttcttg acgttgtctc tggagatggt gaatcggccc ttcacagcgt cagtgtagta 120tgtgctacct ccactgtcac taatatctgc gacccactgc agccccttcc caggagcctg 180gcggatccag ctcatgtagt agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttca ccagttctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 31 vh31acccgcacag taatacacgg cggtgtcctc ggctctcagg ctgttcatct gcagatatag 60cgtgttcttg acgttgtctc tggagatgat gaatcgaccc ttcacagcat ctgcggagct 120tgtgctactt ccaccactgt taatgtatgc gacccactgc acccccttcc ctggagcctg 180acagacccat tgcatgctgt agctactgaa ggtgaatcca taggccacac aggagagtgt 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cccc 294 //SEQ ID NO. 32 vh32tcccacacag taatatacgg ccgtgtcctc ggctctcagg cagttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagta 120tgtgctactt ccaccactgt taatgtatgc aacccactgc aaccctttcc ctggagcctg 180gccgacccag ctcatccaat agctactgaa ggtaaatcca gaggccacac agaagagtct 240cagggacccc ccaagcttca ccaggtctcc cccagcctcc accagctgca tctc 294 //SEQ ID NO. 33 vh33acccgcacag taatacatgg ccgtgtcctc ggctctcagg ctgttcatct gaagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaaccggccc ttcacagcat ctgcatagta 120tgtgctactt ccaccactgc taatgtatgc gacccactgc agccccttcc ctggagcctg 180gcggacccag ttcatgtcat agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggatccc acaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 34 vh34attcacacag taatacatgg ccatgtcctc agctctcagg ctgttcatct gcagatacag 60catgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120catgttactt ccactagaat aaattcatgc gactcacggc agccccttcc caggagcctt 180gtggacccag ctcatggtat aggaaatgaa ggtgaatcca gaggccacac gggcgagtct 240cagggacctc tcaggcttca ccaggtctcc cccagactcc gccagctgcc cctc 294 //SEQ ID NO. 35 vh35 pseudoattcacacag taatacatgg ccatgtcctc agctctcagg ctgttcatct gcagatacag 60catgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120catgttactt ccactagaat aaattcatgc gactcacggc agccccttcc caggagcctt 180gtggacccag ctcatggtat aggaaatgaa ggtgaatcca gaggccacac gggcgagtct 240cagggacctc tcaggcttca ccaggtctcc cccagactcc gccagctgcc cctc 294 //SEQ ID NO. 36 vh36gtctgcacag taatatatgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg acattgtctc tggagatggt gaatcggccc ttcacagtgt ctgcatacct 120tgagatactt ccatcatacc taatccatga gacccactgc agcccctgcc ctggagcctg 180gtggacccag ctcatttcac tgctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggaccct ccaggcttca ccaaatcttc cccagactcc accagctgta cctc 294 //SEQ ID NO. 37 vh37cttcgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagta 120tgtgctactt ccatcattcc aaataactgc gacccactgc agccccttcc ctggagactg 180acggacccag ctcatgtcat agctactaaa ggtgaatcca gaggccacac aggacagtct 240caaggtcccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 38 vh38cttcgcacag taatacacgg ctgtgtcctc ggctctcagg ctgttcatct gaagatacag 60cttgttcttg gcgttctctc tggagatggt gaaccggccc ttcacagcgt ctgcgtagcc 120tgtgctacct ccactatcac taatatctgc gacccactgc agccccttcc caggagcctg 180gcggatccag ctcatgtagt agctactgaa ggtgaatccc gaggccacac aggagagtct 240cagggaaccc ccaggcttca cgaggtctcc cccagattcc accagctgta cctc 294 //SEQ ID NO. 39 vh39cttcgcacag taatacacag ccgtgtcctc ggctctcagg ctgttcatct gcagatacac 60tgtgttcctg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt cagtgtagta 120tgtgctactt ccatcatagc taatagctgc gacccactgc agccccttcc caggagcctg 180gcggacccag ctcatgtcgt agttactgaa ggtgaatcca gaggctacac aggagagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 40 vh40cttcccacag taatacacag caatatcctc agctctcagg ctgttcatct gcagatacac 60catgttcttg gcgttgtcta tggagatggt gaatcggccc ttcacagcat ctacgtagta 120tgtgctactt ccactactgc taatagctgc aacccactgc aggcccttcc ctggagcctg 180gccgacccag ctcatggcat acctactgaa ggtgaatcca gaggtcacac aggagagtct 240cagggacccc cctggcttca ccaggtcttc ccccagactc caccagctgc actt 294 //SEQ ID NO. 41 vh41 pseudoactcgcacag taatacacgg ccgtgtcctc agctctcaag ctgttcatct gcaggtacag 60cgtgttcttg ccattgtctc tggagatggt gaatcggccc ttcacagtgt ctgtgtagta 120tgtgctcctt ccatcatagc taatagctgc gacccactgc agccccttcc ctgaagcctg 180gcagacccag ctcatgccat agctcctgaa ggtgaatcca gaggccacac aggacagtct 240cagggaaccc ccaggcttca tcaggtctcc cccagactcc accagctaca cctt 294 //SEQ ID NO. 42 vh42cctcgcacag taatacacgg ccgtgtcttc ggctctcagg ttgttcatct gcagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcgaccc ttcacagcgt ctgtgtacca 120tgtgctactt ccaccactgt taatgtacgc gacccactgc agctccttcc ctggagcctg 180gcggacacag ctcatccaat agctactgaa ggtgaatcca gaagtcacac atgagagtct 240cagggaaccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 43 vh43atctgcacag taatacaggg ctatgtcctg agctctcagg ctgttcatct gtagataaag 60catgttcttg gctttgtctc tggagatggt gaatcgaccc ttcacagcgt ctgcatagta 120tgtgctgctt ccattactgc taatgtatgt gacccactgt agccccatcc caggagactg 180acggagacaa tgcatgctgt agctactgaa ggtgaacctt gaggccacac aggagagtct 240ctgggacccc ccaggtttca ctcggtctcc cccagactcc agcagctgta cttc 294 //SEQ ID NO. 44 vh44gtctgcacag taatatatgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagtgt ctgcatacct 120tgagctactt ccatcatacc taatccatga gacccactgc agcccctgcc ctggagcctg 180gtggacccag ctcatttcac tgctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggaccct ccaggcttca ccaaatcttc cccagactcc accagctgta cctc 294 //SEQ ID NO. 45 vh45gtccacacag taatacacag ctgtgtcctc ggctctcagg ctgttcatct gcagatacac 60tgtgttcctg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt cagtgtagta 120tgtgctactt ccatcatagc taatagctgt gacccactgc agccccttcc caggagcctg 180gcggacccag ctcatgctgt agctactgaa ggttaatcca gaggccacac aggagagtct 240cagggacccc ccaggcttcg ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 46 vh46 pseudogtgtgcacag taatacacag ccgtgtcctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcat ctgcgtggta 120tgtggtactt ccatctttgt taattactga gactcagtgc agccctttcc ctggagcttg 180gcggacccag cttatccagt aactactgaa agtgaatgca gaggctacaa aggagagtct 240cagggacccc ccaggcttca ccagctctcc cccagattcc accagtggca cctc 294 //SEQ ID NO. 47 vh47 pseudocctcgcacag caatgcacgg ctgtgtcctc agctctcagg ctgttcatct gcagatacag 60catgttcctg gtgctgtctc tggagatggt gaatcagccc tttacagcgt ctgcatagta 120catgctactt ccattactgc tattggatat gacccactgt agccactacc gggagactgg 180tggagccaat gtatgctgtt gctatgaaag gtgaatgtag aggactcaca gaagagtctc 240agggacccgc caggcttcac caggtctccc ctagatttca ccaactgctc ctca 294 //SEQ ID NO. 48 vh48 pseudotccctcgcac agtaatacat ggccttgtcc tcagctctca ggctgttcat ctgcagaaac 60actgtgttct tggcgttctc tggagatggt gaatcggccc ttcacagcat ctgtgtagct 120tgtgctattt ccactagctt aaatccatgc gacccactga atccccttcc cgggagcctg 180gcggacccag ctcatgctgt agctactgaa ggtgaatcca gcggccacac aggagagtct 240cagggacccc ccaggcttca tgaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 49 vh49cttcgcacag taatacacgg ctgtgtcctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcat ctgcgtagcc 120tgtgctcctt ccatcattgc taatccgtgt gagccactgc agccccttcc ctggagcctg 180gcggacccag tccatgtcgt tgctactgaa ggtgaaacca gaggccacac aggagagtct 240caaggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 50 vh50 pseudocttcgcacag taatacatgg ccgtgtcctc agctctcagg ctgttcatct gtagatacag 60catgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagtt 120tgtgctactt ccatccctgc taataactgc gacccactgc agccccttcc ctggagcctg 180gcggacccag tgcatggcat agctactgaa agtgaatccg gaggtcacac aggacagtct 240tagggaaccc caggcttcac caggtctccc ccagactcct ccagctgcac ctca 294 //SEQ ID NO. 51 vh51 pseudocatcgcacag taatataggg ccgtgtcctc ggctctcagg ctggtcatct gcagatacag 60agtgttcttg gaattgtctc tggagatggt gaatcggccc ttcacagcat cagtgtagta 120tgtgctactt ccatcactcc taattcatgc gaaccactgc agccccttcc ctggagcctg 180gtggacccag tgcatgtagt agatactgaa ggtgaatccg gaggccacac aggacagtct 240cagggacccc ccaggcttca ccagctctcc cccatactcc accagctgca cttc 294 //SEQ ID NO. 52 vh52 pseudoactcacacag taatacacgg ccgtgtcttc ggctctcagg ctgttcacct gcaggtacag 60cgtgttcttg gcattgtctc tggagatggt gaatgggccc ctcacagcgt ctgcatagtt 120tgtgctactt ccagtactgc taatttctgc aagccactgc agccccatcc ctggagcctg 180gtggacccag tacatccagt agctactgaa ggtgaatcca gggtccacac aggagagtct 240cagggacccc ccaggcttca ccaggcctcc ctagactcca ccagctgcat ctca 294 //SEQ ID NO. 53 vh53cttcacacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcatagct 120tgtgctactt ccatcactgc taatgtatcc gacccactgc agccccttcc ctggagcctg 180gcggacccag ttcatgtagt tgctactgat agtgaatccg gaggccacac aggagagtct 240cagggacccc ccaggcttca tcaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 54 vh54 pseudoactcacacag taatacgcgg tcatgtcctg ggctctcagg ctgttcatct gcagatacag 60catgttcttg gcattgtctc tgaagatggt gaattggccc ttcactgtgt ctgcatagta 120tgtgctacct ccaccactgt taataattgc aacccacccc aaccctttcc ctggagcctg 180gcagacccaa tgcatccagt agctactgaa ggtgaatcca gaggccacac aagagagtct 240cagagactcc ccaggcttca ccaggtttcc ccagactcca ccagctgcac ctca 294 //SEQ ID NO. 55 vh55 pseudogggaggtttg tgtctgggct cacacttagg tcacctcact gtgtccttcg cacagtaata 60cacggccgtc ttggcattgt ctctggagat ggtgaatcgg cccttcacag cgtctgtgta 120gtatgtgcta cttccgtcac tgctaatccg tgcgacccac ttcagcccct ccctggagcc 180tggcagaccc attccatata gtagctactg aaggtgaatc cagaggccac acaggagagt 240ctcagggatc ccccaggctt caccaggtct cccccagatt ccaccagctg cacctc 296 //SEQ ID NO. 56 vh56 pseudoccttgcacag taatataggg ccgtatcatc agctctcagg ctgttcatct gcagatacag 60agtgttcttg gaattgtctc tggagatggt gaatctgccc ttcacagcgt ctgggtagct 120tgtgctactt ccatcactcc taattcttgc aacccactgc agccccttcc ctggagcctg 180cagacccagt gcatgtagta gctactgaag gtgaatccag aggccacaca ggacagtctc 240agggaacccc caggcttcac cagctctccc ccagactcca caagctgcac ttca 294 //SEQ ID NO. 57 vh57cttcgcacag taatacacgg ctgtgtcctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagtgt ctgcggagcc 120tgtgctactt ccactactgc taataactgc tacccacttc agccccttcc ctggagcctg 180gcggacccag ctcttggcat ggctactgaa ggtgaatccg gaggccacac aagagagtct 240cagggatccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 58 vh58ccgcaatgtg tcactcacac aataatacag ggctatcagg ctgttcatat gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaattgcccc ttcacagcat ctgcatagct 120tttgctgctt ccaccagtat taacccatgt gacccactgc agccccttcc ctggagcctg 180gctgacccag ctcatccagt agctcctgaa ggtgaatcca gaggtcacat aggagagttt 240aattgatccc ccaggcttca acaggtctcc cccagactcc accagcttca cctc 294 //SEQ ID NO. 59 vh59cctcgcacag taatacacgg gcatgtcctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt cagtgtagta 120tgtgctactt ccatcagtgg taatagctgc gacccactgc agccccttcc caggagcctc 180gtggacccag taaatggcat agctatagaa ggttaatcca gaggccacac aggacagtct 240cagggaaccc ccaggcttca ccaggtttcc accagactcc accagctgca cctc 294 //SEQ ID NO. 60 vh60 pseudogtctgcacag taatacagag ccgtgtcctc agctctcagg ctgttcatct gaagatacag 60catgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctttgtaacc 120tgtgctactt ccatcatagc taatgtatgc aacacactgc agccctttcc tggagcctaa 180tggacccagc tcatgtcaca gttactgaag gtgaatcaag gggccacaca ggagagtctc 240agggaccccc cagacttcac cagatctccc cgagactcca ccagctgctc ctca 294 //SEQ ID NO. 61 vh61 pseudocctcgcacag caatgcacgg ctgtgtcctc agctctcagg ttattcatct gcagatacag 60catgttcctg gtgttgtctc tggagatggt gtatcagccc tttacagcgt ctgcatagta 120catgctactt ccattactgc tattggatat gacccactgt agccactacc gggagactgg 180tggagccaat gcatgctgta gctatgaaag gtgaacgtag aggactcaca gaagagtctc 240agggacccgc caggcttcac caggtctccc ctagatttca ccaactgctc ctca 294 //SEQ ID NO. 62 vh62 pseudogtgtcccctg cacaggaata catggccgtg tcctcagctc tcaggcatct gcagaaacag 60tgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagct 120tgtgctactt ccgctagcat atatccatgc gacccactgc atccccttcc cgggagcctg 180gcggacccag ctcatgctgt agctactgaa ggtgaatcca gcggccacac aggagagtct 240cagggacccc ccaggcttca cggggtctcc cgcagactcc accagctgca cctc 294 //SEQ ID NO. 63 vh63 pseudogtctgcacag taatacaagg ccgtgtcctc ggctctcagg ctgttcatct gcagatagag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcat ctgtgtagta 120tgtgctactt ccactattgc taatttttgc gacccactgc agcccttccc aggagcctgg 180cggacacagc tcatgctgta gctactgaag tgaatccaga ggccatacag gacagtctca 240gggaccgccc aggcttcacc aggtctccgc cagactccac cagctgcacc tcac 294 //SEQ ID NO. 64 vh64ctttgcacag taatacatag ccgtgtcctc ggctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagta 120tgtgctactt ccatcactgc taatccgtgc gacccactga agccccttcc ctggagcctg 180gcggacccag tacatgtagt agctactgaa ggtgaatcca gaggccacac aggacagtct 240cagggacccc ccaggcttca ccaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 65 vh65 pseudottgcacaggg atatagggcc gtgtccttgg ctctcaggct gttcatctgc agatacagaa 60tgttcttgga attgtctttg gagatggtgt atcggtcctt cacagcatca gtgtatatgt 120gctacttcca tcactcctaa ttcatgtgac ccacagcagc catttccctg gagcctggcg 180gacccagtac atgtagtagc tactgaaggt gaatccagag gccacacagg agagtctcag 240ggacccccca ggcttcacca gctctccccc agactccacc agctgcactt c 291 //SEQ ID NO. 66 vh66cttcggacag taatacacgg ctgtgtcctc ggatctcagg ctgttcatct gcagatacag 60tgtgttcctg gcattgtctc tggagatggt gaaccggccc ttcacagcat ctgcgtagta 120tgtgctactt ccaccactgt taatgtatgc gacccactgc agccccttcc ctggagcctg 180gcggacccag ctcatggcat agctactgaa ggtgaatcca gaggccacac aggacagtct 240cagggatccc ccaggcttca ccaggtctcc cccagactcc accagctgta cctc 294 //SEQ ID NO. 67 vh67ccttgcacag taatatacag ccgtgtcctc ggctctcagg ctgtgcatcc gtagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctgcgtagct 120tgtgctattt ccactactgc taatttctgc aacacactgt agccccttcc ctggagcctg 180gcagaaccag ctcatgtaga agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggaccgc tcaggcttca caaggtctcc tccagactcc accagctgca cctc 294 //SEQ ID NO. 68 vh68ccttgcacag ttatacaggg ccgtatcctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggaaatggt gaatcggccc ttcacagcgt ctgcgtatta 120tgtgctactt ccatcactgc taatgtatcc gacccactgc agccccttcc taggagcctg 180gcggacccag ctcatggcat agctactgaa ggtgaatcca gaggccacac aggacagtct 240cagggaatcc ccaggcttca ccaggtctcc gccagactcc accagctgca cctc 294 //SEQ ID NO. 69 vh69 pseudogtctgcacag taatacaggg ccgtgtgctc agctctcagg ctgttcatct gaagatacag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc tgcacagcgt cifigtaacc 120tgtgctactt ccatcatagc taatatatgc aatacactgc agccctttcc tggagtctaa 180tggaccgagc tcatgtcata gttactgaag gtgaatccag gggccacaca ggagagtctc 240agggaccccc caggcttcac caggtctccc ccagactcca tcagctgcac ctca 294 //SEQ ID NO. 70 vh70 pseudoccctcgcaca gtaatacaca gcggtgtcct cggctctcag gctgttcaac tgcagataca 60gcatgttctt ggcgttgtct ctggagaggt gaatcggccc ttcacagcat ctgtgtacct 120tgtgctactt ccaccactgt taatgtatgc gacccactgc agccacttcc ctggagcctg 180gcggacccag ctcatgatgt agctactgaa ggtgaatcca gaagccacac aggagagtct 240caggagccca caaggcttca ccaagtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 71 vh71gtctgcacag taatacacgg ccgtgtcctc ggctctcagg ctgttcatct gcagatacac 60tgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcgt ctacatggta 120tgtgctactt ccactactgc taatagctgc aacccactgc agccccttcc ctggagcctg 180gcagacccaa ctcatggcat agctactgaa ggtgaatcca gaggccacac aggagagtct 240cagggacccc cctggcttca ccagatctcc cccaagctcc accagctgca attc 294 //SEQ ID NO. 72 vh72gtgtgcacag taatacacag tcgtgccctc agctctcagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaatcggccc ttcacagcat ctgcgtggta 120tgtggtactt ccatctttgt taattactga gacccagtgc agccctttcc ctggagcttg 180gcggacccag cttatccagt aactactgaa agtgaatcca gaggctacac aggagaatct 240cagggacccc tcaggcttca ccagctctcc cccagattcc accagtggca cctc 294 //SEQ ID NO. 73 vh73pseudoccttgcacag gaatacatgg ccgtgttctc agctctcagg ctgttcatct gcagaaacac 60tgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcat ctgtgtagct 120tgtgctactt ccactagcat aaatctatgt gacccactgc acccccttcc cgggagcctg 180gcggacccag ctcatgctgt agctactaaa ggtgaatcca gcggccacac aggagagtct 240cagggacccc tccggcttca caaggtctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 74 vh74ctttgcacag taatacacgg ccgtgtcctc ggctcccagg ctgttcatct gcagatacag 60cgtgttcttg gcattgtctc tggagatggt gaattgaccc ttcacagcgt ctgggtagta 120tgtgctactt ccatcactgc taatctgtgc gacccactgc agccccttcc ctggagcctg 180gcggacccat tccatgtagt agctactgaa ggtgaatcca gaggccacac aagagagtct 240cagggatccc ccaggcttca ccagatctcc cccagactcc accagctgca cctc 294 //SEQ ID NO. 75 vh75catcgcacag taatacacag cagagtcctc ggctctcagg ctgttcatct gcagatagag 60cgtgttcttg gcgttgtctc tggagatggt gaatcggccc ttcacagcct gtgggtagta 120tgtgctactt ccatcactgc taatccctgc gacccactgc agcctcttcc ctggagcctg 180gcggacccag tccatgtagt agctactgaa ggtgaatcca gaggccacac aagagagtct 240cagggacccc ccaggcttca ccaggtttcc tccagactcc accagctgca cctc 294 //SEQ ID NO. 76 vh76ccttgcacag taatataggg ccatgtcctc ggctctctgg ctgttcatct gcagatacag 60cgtattcttg gaattgtctc tggagatggt aaattggccc ttcatggcgt ctgcgtacct 120tgtgctactt ccatcattgc taatccttgt gacccactgc agccccttcc ctgtagcctg 180gcggacccag tgcatgtagt agctactgaa ggtgaatcca gaggccacac aagagagtct 240cagggacccc ccagacttca ccaggtctcc tccagactcc accagctgca cctc 294 //SEQ ID NO. 77 vh77cttcgcacag taatgcatgg ccctgtcctc agctctcagg ctgttcatct gcagatagag 60cgtgttcttg gcgttttctc tggagatggt gaatcggccc ttcactgtgt ctgcgtagta 120tatgctacct ccaccactgt taataattgc gacccactcc agccctttcc ctggagcctg 180gaggacccag tgcatccagt agctactgaa ggtgaatcca gaggacacac aagaaagtct 240cagagacccc ccaggcttca ccaggtctcc cccagactct accagctgca actc 294Dh sequences Dh1 gtactactgt actgatgatt actgtttcaa c (SEQ ID NO. 78)Dh2 ctactacggt agctactac (SEQ ID NO. 79)Dh3 tatatatata tggatac (SEQ ID NO. 80)Dh4 gtatagtagc agctggtac (SEQ ID NO. 81)Dh5 agttctagta gttggggct (SEQ ID NO. 82)Dh6 ctaactgggg c (SEQ ID NO. 83) JH sequencesJh1 tgaggagacg gtgaccaggg tgccctggcc ccagaggt ctaagta (SEQ ID NO. 84)Jh2 tgaggagaca gtgaccaggg tgccctggcc ccagtaac caaa (SEQ ID NO. 85)Jh3 tgaggagacg gtgaccaggg ttccctggcc ccagtagt caaa (SEQ ID NO. 86Jh4 tgaggacaca gtgaccaggg tcccttggcc ccagtagt (SEQ ID NO. 87)Jh5 tgaggacacg aagagtgagg tgccatggcc ccagtagt ccatacc atagtaat(SEQ ID NO. 88)

TABLE 2 Canine Igκ Sequence Information Vκ genes SEQ ID NO. 89: vk1  1 GACATCACGA TGACTCAGTG TCCAGGCTCC CTGGCTGTGT CTCCAGGTCA 51 GCAGGTCACC ACGAACTGCA GGGCCAGTCA AAGCGTTAGT GGCTACTTAG101 CCTGGTACCT GCAGAAACCA GGACAGCGTC CTAAGCTGCT CATCTACTTA151 GCCTCCAGCT GGGCATCTGG GGTCCCTGCC CGATTCAGCA GCAGTGGATC201 TGGGACAGAT TTCACCCTCA CCGTCAACAA CCTCGAGGCT GAAGATGTGA251 GGGATTATTA CTGTCAGCAG CATTATAGTT CT SEQ ID NO. 90: vk2  1 GATATTGTCA TGACACAGGC CCCACCGTCC CTGTCCGTCA GCCCTGGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAATG101 GGAACACCTA TTTGTATTGG TTCCGACAGA AGCCAGGCCA GTCTCCAGAG151 GGCCTGATCT ATAAGGTGTC CAACCGCTTC ACTGGCGTGT CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TGCTGGAGTT TATTACTGCG GGCAAAATTT ACAGTTTSEQ ID NO. 91: vk3  1 ATTGTCATGA CACAGACGCC ACCGTCCCTG TCTGTCAGCC CTAGAGAGAC 51 GGCCTCCATC TCCTGCAAGG CCAGTCAGAG CCTCCTGCAC AGTGATGGAA101 ACACCTATTT GGATTGGTAC CTGCAAAAGC CAGGCCAGTC TCCACAGCTT151 CTGATCTACT TGGTTTCCAA CCGCTTCACT GGCGTGTCAG ACAGGTTCAG201 TGGCAGCGGG TCAGGGACAG ATTTCACCCT GAGAATCAGC AGAGTGGAGG251 CTAACGATAC TGGAGTTTAT TACTGCGGGC AAGGTACACA GCTT SEQ ID NO. 92: vk4  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAATG101 GGAACACCTA TTTGTATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGATGA TGCTGGAGTT TATTACTGCG GGCAAGGTAT ACA SEQ ID NO. 93: vk5  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCTGTCA GCCCTGGAGA 51 GACTGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTGATG101 GAAACACGTA TTTGAACTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTAATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGCG GGCAAGGTAT ACA SEQ ID NO. 94: vk6  1 GATATTGTCA TGACACAGAA CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GACGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAACG101 GGAACACCTA TTTGAATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 GGCCTGATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TGCTGGAGTT TATTACTGCA TGCAAGGTAT ACA SEQ ID NO. 95: vk7  1 ATTGTCATGA CACAGACCCC ACCGTCCCTG TCCGTCAGCC CTGGAGAGCC 51 GGCCTCCATC TCCTGCAAGG CCAGTCAGAG CCTCCTGCAC AGTAACGGGA101 ACACCTATTT GAATTGGTTC CGACAGAAGC CAGGCCAGTC TCCACAGGGC151 CTGATCTATA GGGTGTCCAA CCGCTCCACT GGCGTGTCAG ACAGGTTCAG201 TGGCAGCGGG TCAGGGACAG ATTTCACCCT GAGAATCAGC AGAGTGGAGG251 CTGACGATGC TGGAGTTTAT TACTGCGGGC AAGGTATACA SEQ ID NO. 96: vk8  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCTGTCA GCCCTGGAGA 51 GACTGCCTCC ATCTCTTGCA AGGCCAGTCA GAGCCTCCTG CACAGTGATG101 GAAACACGTA TTTGAATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGCG GGCAAGTTAT ACA SEQ ID NO. 97: vk9  1 ATTGTCATGA CACAGACCCC ACTGTCCCTG TCCGTCAGCC CTGGAGAGAC 51 TGCCTCCATC TCCTGCAAGG CCAGTCAGAG CCTCCTGCAC AGTGATGGAA101 ACACGTATTT GAATTGGTTC CGACAGAAGC CAGGCCAGTC TCCACAGCGT151 TTGATCTATA AGGTCTCCAA CAGAGACCCT GGGGTCCCAG ACAGGTTCAG201 TGGCAGCGGG TCAGGGACAG ATTTCACCCT GAGAATCAGC AGAGTGGAGG251 CTGACGATAC TGGAGTTTAT TACTGCATGC AAGGTACACA GTTT SEQ ID NO. 98: vk10  1 TCGTCATGAC ACAGACCCCA CTGTCCCTGT CCGTCAGCCC TGGAGAGACT 51 GCCTCCATCT CCTGCAAGGC CAGTCAGAGC CTCCTGCACA GTAACGGGAA101 CACCTATTTG TTTTGGTTCC GACAGAAGCC AGGCCAGTCT CCACAGCGCC151 TGATCAACTT GGTTTCCAAC AGAGACCCTG GGGTCCCACA CAGGTTCAGT201 GGCAGCGGGT CAGGGACAGA TTTCACCCTG AGAATCAGCA GAGTGGAGGC251 TGACGATGCT GGAGTTTATT ACTGCGGGCA AGGTATACA SEQ ID NO. 99: vk11  1 GATATTGTCA TGACACAGGC CCCACCGTCT CTGTCCGTCA GCCCTAGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAATG101 GGAACACCTA TTTGTATTGG TTCCGACAGA AGCCAGGCCA GTCTCCAGAG151 GGCCTGATCT ATAAGGTGTC CAACCGCTTC ACTGGCGTGT CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TGCTGGAGTT TATTACTGCG GGCAAGGTAT ACAGTTTSEQ ID NO. 100: vk12  1 GATATCGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAACG101 GGAACACCTA TTTGTTTTGG TTTCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC ACTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCACAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGCG GGCAAGGTAC ACAGTTTSEQ ID NO. 101: vk13  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCTGTCA GCCCTGGAGA 51 GACTGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTGATG101 GAAACACGTA TTTGAATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC ACTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGTG GGCAAGTTAT ACA SEQ ID NO. 102 vk14  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GACTGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTGATG101 GAAACACGTA TTTGAATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC ACTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCACAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGCG GGCAAGGTAC ACAGTTTSEQ ID NO. 103: vk15  1 GATATTGTCA TGACACAGAA CCCACTGTCC CTGTCTGTCA GCCCTGGAGA 51 GACGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTGATG101 GAAACACGTA TTTGAACTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTAATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TACTGGAGTT TATTACTGCG GGCAAGGTAT ACA SEQ ID NO. 104: vk16  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAATG101 GGAACACCTA TTTGTATTGG TTCCAACAGA AGCCAGGCCA GTCTCCACAG151 CGTTTGATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGATGA TGCTGGAGTT TATTACTGCG GGCAAGGTAT ACA SEQ ID NO. 105: vk17  1 GATATTGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GACGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAACG101 GGAACACCTA TTTGAATTGG TTCCGACAGA AGCCAGGCCA GTCTCCACAG151 GGCCTGATCT ATAAGGTCTC CAACAGAGAC CCTGGGGTCC CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TGCTGGAGTT TATTACTGCA TGCAAGGTAT ACA SEQ ID NO. 106: vk18  1 GATATCGTCA TGACACAGAC CCCACTGTCC CTGTCCGTCA GCCCTGGAGA 51 GCCGGCCTCC ATCTCCTGCA AGGCCAGTCA GAGCCTCCTG CACAGTAATG101 GGAACACCTA TTTGTATTGG TTCCGACAGA AGCCAGGCCA GTCTCCAGAG151 GGCCTGATCT ATAAGGTGTC CAACCGCTTC ACTGGCGTGT CAGACAGGTT201 CAGTGGCAGC GGGTCAGGGA CAGATTTCAC CCTGAGAATC AGCAGAGTGG251 AGGCTGACGA TGCTGGAGTT TATTACTGCG GGCAAGGTAT ACASEQ ID NO. 107: vk19 Pseudogene  1 TCTTGACCTA GTCTCCAGCC TCCCTGGCTA TTTCCCAAGG GGACAGAGTC 51 AACCATCACC TATGGGACCA GCACCAGTAA AAGCTCCAGC AACTTAACCT101 GGTACCAACA GAACTCTGGA GCTTCTTCTA AGCTCCTTGT TTACAGCACA151 GCAAGCCTGG CTTCTGGGAT CCCAGCTGGC TTCATTGGCA GTGGATGTGG201 GAACTCTTCC TCTCTCACAA TCAATGGCAT GGAGGCTGAA GGTGCTGCCT251 ACTATTACTA CCAGCAGTAG GGTAG Jκ genesJκ1 gtggacgttc ggagcaggaa ccaaggtgga gctcaaac (SEQ ID NO. 108)Jκ2 ttatactttc agccagggaa ccaagctgga gataaaac (SEQ ID NO. 109)Jκ3 gttcactttt ggccaaggga ccaaactgga gatcaaac (SEQ ID NO. 110)Jκ4 gcttacgttc ggccaaggga ccaaggtgga gatcaaac (SEQ ID NO. 111)Jκ5 gatcaccttt ggcaaaggga cacatctgga gattaaac (SEQ ID NO. 112)

TABLE 3 Canine Igλ sequences Vλ germline genes VL1 (SEQ ID NO. 113)tcctcttgtc taaagaaaag aacatcactc tctctgtgtc tctccccctt tcagggtcctgggaccagtc tgtgctgact cagccgccct cagtgtcggg atctgtgggc cagagaatcaccatctcctg ctctggaagc acaaacagct accaacagct ctcaggaaag gcctctaaactcctcgtaga tggtactggg aaccgaccct caggggtccc cgaccgattt tctggctccaaatctggcaa ctcaggcact ctgaccatca ctgggcttgg gacgaggctg aggacgaggctgaggacgag gctgattatt attgttagtc cactgatctc acgcttggtg ctcccacagtgctctgggcc tacggggaag tgagacacaa acctgctgtc cctagaacaa tggcactgcctgtgcaaccc tggccttagg VL2 pseudo (SEQ ID NO. 114)cagtctgtac tgactcagcc ggcctcagtg tctgggtccc tgggccagag ggtcaccatctcctgcactg gaagcagctc caacatcggt ggatattatg tgagctggct ctagcagctcccgggaacag gccccagaac catcatctat agtagtagta accgaccttc aggggtccctgatcgattct ctggctccag gtcaggcagc acagccaccc tgaccatctc tgggctccaggctgaggatg aggctgatta ttactgttca acatacgaca gcagtctcaa agctcccacagtgctccagg cctgtgggga agtgagacaa aaacccattt acctatctgc aatgtgagtgagcgccccag gagcttcctg cgtaggctcc cctgggtttc tgctgattct tcagttgatgccctgagccc aggtg VL3 pseudo (SEQ ID NO. 115)atcccaggct gtggtgaccc agcttccttc tctgcatccc tgggaacaac agccagactcacatgcaccc tgagctgtgg cttcagtatt gatagatatg ctataaactg gttccagcagaaggcagaga gccttccctg gtacctactg tgctattact ggtactcaag tacacagttgggcttcagcg tccccagctg catctctgga tccaagacaa ggccacattc acaaacgagtagacccatct ctggttgggt ctagagctcc agccccacct gagactgatg cacaattg VL4(SEQ ID NO. 116)ggcccaggct gtgctgactc agctgccctc agtgtctgca gccctgggac agagggtcaccatctgcact ggaagcagca ccaacatcgg cagtggttat tatacactat ggtaccagcagctgcaggaa agtcccctaa aactatcatc tatggtaata gcaatcgacc cttgagggtcccggatcgat tctctggctc caagtatggc aattcagcca cgctgaccat cactgggctccaggctgagg acgaggatga ttattactgc cagtcctctg atgacaacct VL5(SEQ ID NO. 117)cagtctgtgc tgactcagcc ggcctcggtg tctgggtccc tgggccagag ggtcaccatctcctgcactg gaagcagctc caatgttggt tatggcaatt atgtgggctg gtaccagcagcttccaggaa caggccccag aaccattatc tgttatacca atactcgacc ctctggggttcctgatcgat actctggctc caagtcaggc agcacagcca ccctgaccat ctctgggctccaggctgaag acgagactga ttattactgt actacgtgtg acagcagtct caatgctagcacagtgctcc aggcctttgg agag VL6 pseudo (SEQ ID NO. 118)gtgatggtga gggcgacttt gttcccagag atggatccag agaagcgatc agggaccccagaagggtgtc tgcttgtgct gtagataagc atgcaaggag cctggccttg ggtctgctggtaccagctgg ggtagtttct tgtagagact tacccagagc tgaggccaca tgtgaatgtgactgtccctc ctggagacac tgagagtgac ggatcctggg tgaccacagt ctg VL7(SEQ ID NO. 119)cagactgtgg taacccagga gccatcactc tcagtgtctc caggagggac agtcacactcacatgtggcc tcagctctgg gtcagtctct acaagtaatt accctggctg gtaccagcagacccaaggcc gggctcctcg cacgattatc tacaacacaa gcagccgccc ctctggggtccctaatcgct tctctggatc catctctgga aacaaagccg ccctcaccat cacaggagcccagcccgagg atgaggctga ctattactgt tccttgtata cgggtagtta c VL8(SEQ ID NO. 120)cagtctgtgc tgactcagcc tccctcagtg tccgggttcc tgggccagag ggtcaccatctcctgcactg gaagcagctc caacatcggt agaggttatg tgcactggta ccaacagctcccaggaacag gccccagaac cctcatctat ggtattagta accgaccctc aggggtccccgatcgattct ctggctccag gtcaggcagc acagccactc tgacaatctc tgggctccaggctgaggatg aggctgatta ttactgctca tcctgggaca gcagtctc VL9 pseudo(SEQ ID NO. 121)cagcctgtga tgacccagct gtcctccctc tctgcatccc tggaaacaac aaccagacacacctgcaccc tgagcagtgg cttcagaaat aacagctgtg taataagttg attccagcagaagtcaggga gccctccctg gtgtctcctg tactattact cagactcaag tatacatttgggctctgagg ttcccagctg cttctctgga tccaagacaa ggccacaccc acactgagtagacccatccc tgggtgggtc tagagctcca gccccactgg aggctgatgc acaattgca VL10(SEQ ID NO. 122)ctgactcaaa cggcctccat gtctgggtct ctgggccaga gggtcaccgt ctcctgcactggaagcagtt ccaacgttgg ttatagaagt tatgtgggct ggtaccagca gctcccaggaacaggcccca gaaccatcat ctataatacc aatactcgac cctctggggt tcctgatcgattctctggct ccatatcagg cagcacagcc accctgacta ttgctggact ccaggctgaggacgaggctg attattactg ctcatcctat gacagcagtc tc VL11 pseudo(SEQ ID NO. 123)cagtctgtgc tgaatcagct gccttcagtg ttaggatccc tgggccagag aatcaccatctcctgctctg gaagcacgaa tgacatcggt atgcttggtg tgaactggta ccaagagccgccaggaaagg cccctaaact cctcgtagat ggtactggga atcgaccctc agggtccctgccgattttct ggctccaaat ctggcaactc aggcactctg accatcactg ggctccaggctgaggacgag gctgattatt attgtcagtc c VL12 (SEQ ID NO. 124)ctgctgtccc aggatgagca gtaataatca gcctcatcct cagcctggaa cccagagattgtcagagtgt ctgtgctgcc tgacctggag ccagagaatt gattggggac ccctgagggttggttactac taccatatat gagggttctt gggcgtgttc ccaggagctg ttggtaccagatcacataac ctctaccgac gttggagctg cttccagtgc aggatatagt gaccctctggcccagggacc tgaacactga gggaggctga gtcagcacag actg VL13 pseudo(SEQ ID NO. 125)cagtctgtgc tgactcaacc agtctcagtg tctggggccc tgtgccagag ggtcaccatctcctgcactg gaaacagctc caacattggt tatagcagtt gtgtgagctg atatcagcagctcccaggaa caggccccag aaccatcatc tatagtatga atactcaacc ctctggggttcctgatcgat tctctggctc caggtcaggc aactcagcca ccctaaccat ctctgggctccaggctgagg acaaggctga ctattactgc tcaacatatg acagcagtct cagtgctcacacggtgctcc aggcctgtgg ggaattgaga caaaaaccta cttatctgtc tgcagtgagc ggagJλ germline genesJL1 agtgtgttcg gcggaggcac ccatctgacc gtcctcg (SEQ ID NO. 126)Jl2 tacgtgttcg gctcaggaac ccaactgacc gtccttg (SEQ ID NO. 127)JL3 tattgtgttc ggcggaggca cccatctgac cgtcctcg (SEQ ID NO. 128)JL4 tggtgtgttc ggcggaggca cccacctgac cgtcctcg (SEQ ID NO. 129)JL5 tgctgtgttc ggcggaggca cccacctgac cgtcctcg (SEQ ID NO. 130)

TABLE 4 Miscellaneous sequence data. A. Pre-DJThis is a 21609 bp fragment upstream of the Ighd-5DH gene. The pre-DJ sequence can be found in Musmusculus strain C57BL/6J chromosome 12, Assembly:GRCm38.p4, Annotation release 106, Sequence ID: NC_000078.6The entire sequence lies between the two 100 bp sequences shown below:Upstream of the Ighd-5 DH gene segment, corre-sponding to positions 113526905-113527004 in NC_000078.6:ATTTCTGTACCTGATCTATGTCAATATCTGTACCATGGCTCTAGCAGAGATGAAATATGAGACAGTCTGATGTCATGTGGCCATGCCTGGTCCAGA CTTG (SEQ ID NO. 131)2 kb upstream of the Adam6a gene correspondingto positions 113548415-113548514 in NC_000078.6:GTCAATCAGCAGAAATCCATCATACATGAGACAAAGTTATAATCAAGAAATGTTGCCCATAGGAAACAGAGGATATCTCTAGCACTCAGAGACTGA GCAC (SEQ ID NO. 132)B. Adam6a Adam6a (a disintegrin and metallopeptidase domain6A) is a gene involved in male fertility. TheAdam6a sequence can be found in Mus musculusstrain C57BL/6J chromosome 12, Assembly:GRCCm38.p4, Annotation release 106, Sequence ID:NC_000078.6 at position 113543908-113546414.Adam6a sequence ID: OTTMUSG00000051592 (VEGA)

TABLE 5 Chimeric canine/mouse Ig gene sequences. Igk Version ASequence upstream of mouse Igkv 1-133GCATTGAATAAACCAGTATAAACAAGCAAGCAAAGATAGATAGATAGATAGATAGATAGATAGATAGATACATAGATAGATAGATAGATAGATAGATGATAGATAGATAGATAGATAGATAGATTTTTACGTATAATACAATAAAAACATTCATTGTCCCTCTATTGGTGACTACTCAAGGAAAAAAATGTTCATATGCAAGAAAAAATGTTATCATTACCAGATGATCCAGCAATCTAGCAATATATATATTGTTTATTCACAAAACATGAATGAACCTTTTAAGAAGCTGTTACAGTGTAAAAATTAAGTTAAATCACTGAAGAACATATACTGTGTGATTTCATTCAAATGAAATTTGAGAAGTAAATATATATGTATATATATATATATGTAAAAAATATAAGTCTGAACTACAAAAATTCAATTTGTTTGATATGTAAGAATAAGAAAAATTGACCCCCAAAATTTGTTAATAATTAGGTATGTGTATTTTTATGAATATATAAGTATAATAATGCTTATAGTATACACTATTCTGAATCACATTTATTCCCTAAGTGTGTTCCCTTGATTATAATTAAAAGTATATTTTTTAAATACAGAGTCAGAGTACAGTCAATAAGGCGAAAATATAGTTGAATGATTTGCTTCAGCTTTTGTAATGTACTAGAGATTGTGAGTACAAAGTCTCAGAGCTCATTTTATCCCTGACAATAACCAGCTCTGTGCTTCAAGTACATTTCCATCTTTCTCTGAAATTTAGTCTTATATAGATAGACAAAATTTAAGTAAATTTCAAACTACACAGAACAACTAAGTTGTTGTTTCATATTGATAATGGATTTGAACTGCATTAACAGAACTTTAACATCCTGCTTATTCTCCCTTCAGCCATCATATTTTGCTTTATTATTTTCACTTTTTGAGTTATTTTTCACATTCAGAAAGCTCACATAATTGTCACTTCTTTGTATACTGGTATACAGACCAGAACATTTGCATATTGTTCCCTGGGGAGGTCTTTGCCCTGTTGGCCTGAGATAAAACCTCAAGTGTCCTCTTGCCTCCACTGATCACTCTCCTATGTTTATTTCCTCAAA(SEQ ID NO. 133)Canine exon 1 (leader) from LOC475754 (SEQ ID NO. 134):atgaggttcccttctcagctcctggggctgctgatgctctggatccCanine intron 1 from LOC475754 (SEQ ID NO. 135)Caggtaaggacagggcggagatgaggaaagacatgggggcgtggatggtgagctcccctggtgctgtttctctccctgtgtattctgtgcatgggacagattgccctccaacagggggaatttaatttttagactgtgagaattaagaagaatataaaatatttgatgaacagtactttagtgagatgctaaagaagaaagaagtcactctgtcttgctatcttgggttttccatgataattgaatagatttaaaatataaatcaaaatcaaaatatgatttagcctaaaatatacaaaacccaaaatgattgaaatgtcttatactgtttctaacacaacttgtacttatctctca ttattttaggatccagtgggCanine 5′ part of exon 2 (leader) from LOC475754 (SEQ ID NO. 136)aggatccagtggg Canine Vκ from LOC475754 (SEQ ID NO. 137)Gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctccatctcctgcaaggccagtcagagcctcctgcacagtgatggaaacacgtatttgaactggttccgacagaagccaggccagtctccacagcgtttaatctataaggtctccaacagagaccctggggtcccagacaggttcagtggcagcgggtcagggacagatttcaccctgagaatcagcagagtggaggctgacgatactggagtttattactgcgggcaaggtatacaagat Mouse RSS heptamer CACAGTGMouse sequence downstream of RSS heptamer (SEQ ID NO. 138)ATACAGACTCTATCAAAAACTTCCTTGCCTGGGGCAGCCCAGCTGACAATGTGCAATCTGAAGAGGAGCAGAGAGCATCTTGTGTCTGTGTGAGAAGGAGGGGCTGGGATACATGAGTAATTCTTTGCAGCTGTGAGCTCTG Igk version BSequence upstream of mouse Igkv 1-133 (SEQ ID NO. 133)GCATTGAATAAACCAGTATAAACAAGCAAGCAAAGATAGATAGATAGATAGATAGATAGATAGATAGATACATAGATAGATAGATAGATAGATAGATGATAGATAGATAGATAGATAGATAGATTTTTACGTATAATACAATAAAAACATTCATTGTCCCTCTATTGGTGACTACTCAAGGAAAAAAATGTTCATATGCAAGAAAAAATGTTATCATTACCAGATGATCCAGCAATCTAGCAATATATATATTGTTTATTCACAAAACATGAATGAACCTTTTAAGAAGCTGTTACAGTGTAAAAATTAAGTTAAATCACTGAAGAACATATACTGTGTGATTTCATTCAAATGAAATTTGAGAAGTAAATATATATGTATATATATATATATGTAAAAAATATAAGTCTGAACTACAAAAATTCAATTTGTTTGATATGTAAGAATAAGAAAAATTGACCCCCAAAATTTGTTAATAATTAGGTATGTGTATTTTTATGAATATATAAGTATAATAATGCTTATAGTATACACTATTCTGAATCACATTTATTCCCTAAGTGTGTTCCCTTGATTATAATTAAAAGTATATTTTTTAAATACAGAGTCAGAGTACAGTCAATAAGGCGAAAATATAGTTGAATGATTTGCTTCAGCTTTTGTAATGTACTAGAGATTGTGAGTACAAAGTCTCAGAGCTCATTTTATCCCTGACAATAACCAGCTCTGTGCTTCAAGTACATTTCCATCTTTCTCTGAAATTTAGTCTTATATAGATAGACAAAATTTAAGTAAATTTCAAACTACACAGAACAACTAAGTTGTTGTTTCATATTGATAATGGATTTGAACTGCATTAACAGAACTTTAACATCCTGCTTATTCTCCCTTCAGCCATCATATTTTGCTTTATTATTTTCACTTTTTGAGTTATTTTTCACATTCAGAAAGCTCACATAATTGTCACTTCTTTGTATACTGGTATACAGACCAGAACATTTGCATATTGTTCCCTGGGGAGGTCTTTGCCCTGTTGGCCTGAGATAAAACCTCAAGTGTCCTCTTGCCTCCACTGATCACTCTCCTATGTTTATTTCCTCAAAMouse Igkv 1-133 exon 1 (leader) (SEQ ID NO. 139)ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGMouse Igkv 1-133 intron 1 (SEQ ID NO. 140)GTAAGGAGTTTTGGAATGTGAGGGATGAGAATGGGGATGGAGGGTGATCTCTGGATGCCTATGTGTGCTGTTTATTTGTGGTGGGGCAGGTCATATCTTCCAGAATGTGAGGTTTTGTTACATCCTAATGAGATATTCCACATGGAACAGTATCTGTACTAAGATCAGTATTCTGACATAGATTGGATGGAGTGGTATAGACTCCATCTATAATGGATGATGTTTAGAAACTTCAACACTTGTTTTATGACAAAGCATTTGATATATAATATTTTTAAATCTGAAAAACTGCTAGGATCTTACTTGAAAGGAATAGCATAAAAGATTTCACAAAGGTTGCTCAGGATCTTTGCACATGATTTTCCACTATTGTATTGTAATTTCAGMouse Igkv 1-133 5′ part of exon 2 (leader) (SEQ ID NO. 141) AAACCAACGGTCanine Vκ from LOC475754 (SEQ ID NO. 142)Gatattgtcatgacacagaccccactgtccctgtctgtcagccctggagagactgcctccatctcctgcaaggccagtcagagcctcctgcacagtgatggaaacacgtatttgaactggttccgacagaagccaggccagtctccacagcgdtaatctataaggtctccaacagagaccctggggtcccagacaggdcagtggcagcgggtcagggacagatttcaccctgagaatcagcagagtggaggctgacgatactggagtttattactgcgggcaaggtatacaagat Mouse RSS heptamer CACAGTGMouse sequence downstream of RSS heptamer (SEQ ID NO. 138)ATACAGACTCTATCAAAAACTTCCTTGCCTGGGGCAGCCCAGCTGACAATGTGCAATCTGAAGAGGAGCAGAGAGCATCTTGTGTCTGTGTGAGAAGGAGGGGCTGGGATACATGAGTAATTCTTTGCAGCTGTGAGCTCTG

What is claimed is:
 1. A method of producing antibodies comprising fullycanine heavy and/or light chain variable domains and rodent constantdomains, the method comprising: (i) providing a transgenic rodent with agenome in which an entire endogenous immunoglobulin variable gene locushas been deleted and replaced with an engineered partly canineimmunoglobulin locus comprising canine immunoglobulin variable geneV_(H), D and J_(H) and/or canine V_(L) and J_(L) coding sequences androdent immunoglobulin variable gene locus non-coding regulatorysequences, wherein the engineered partly canine immunoglobulin locus ofthe transgenic rodent is functional and expresses immunoglobulin chainscomprised of canine variable domains and rodent constant domains; and(ii) isolating the antibodies comprising canine carriable regions androdent constant regions expressed by the transgenic rodent.
 2. An partof or a whole antibody molecule comprising canine variable domains androdent constant domains produced by the method of claim
 1. 3. The methodof claim 1, wherein the rodent is a mouse or a rat.
 4. The method ofclaim 1, wherein the non-coding regulatory sequences comprise promoterspreceding individual V gene segments, splice sites, and recombinationsignal sequences for V(D)J recombination.
 5. The method of claim 1,wherein the engineered partly canine immunoglobulin locus furthercomprises an ADAM6 gene.
 6. The method of claim 1, wherein theengineered partly canine immunoglobulin locus further comprisesPax-5-Activated Intergenic Repeat (PAIR) elements.
 7. The method ofclaim 1, wherein the engineered partly canine immunoglobulin locusfurther comprises CTCF binding sites from a heavy chain intergeniccontrol region
 1. 8. A method of producing monoclonal antibodiescomprising fully canine variable regions and rodent constant regions,the method comprising: (i) providing a transgenic rodent with a genomein which an entire endogenous immunoglobulin variable gene locus hasbeen deleted and replaced with an engineered partly canineimmunoglobulin locus comprising canine immunoglobulin variable geneV_(H), D and J_(H) and/or canine V_(L) and J_(L) coding sequences androdent immunoglobulin variable gene locus non-coding regulatorysequences, wherein the engineered partly canine immunoglobulin locus ofthe transgenic mouse is functional and expresses immunoglobulin chainscomprised of canine variable domains and rodent constant domains; (ii)obtaining B-cells from the transgenic rodent; (iii) immortalizing theB-cells; and (iv) isolating antibodies comprising canine variabledomains and rodent constant domains expressed by the immortalizedB-cells.
 9. A part of or a whole monoclonal antibody comprising caninevariable domains and rodent constant domains produced by the method ofclaim
 8. 10. The method of claim 8, wherein the rodent is a mouse orrat.
 11. The method of claim 8, wherein the non-coding regulatorysequences comprise promoters preceding individual V gene segments,splice sites, and recombination signal sequences for V(D)Jrecombination.
 12. The method of claim 8, wherein the engineered partlycanine immunoglobulin locus further comprises an ADAM6 gene.
 13. Themethod of claim 8, wherein the engineered partly canine immunoglobulinlocus further comprises Pax-5-Activated Intergenic Repeat (PAIR)elements.
 14. The method of claim 8, wherein the engineered partlycanine immunoglobulin locus further comprises CTCF binding sites from aheavy chain intergenic control region
 1. 15. A B lymphocyte from atransgenic rodent with a genome in which an entire endogenousimmunoglobulin variable gene locus has been deleted and replaced with anengineered partly canine immunoglobulin locus comprising canineimmunoglobulin variable gene V_(H), D and J_(H) and/or canine V_(L) andJ_(L) coding sequences and rodent immunoglobulin variable gene locusnon-coding regulatory sequences, wherein the engineered partly canineimmunoglobulin locus of the transgenic rodent is functional andexpresses immunoglobulin chains comprised of canine variable domains androdent constant domains.
 16. A part of or whole immunoglobulin moleculecomprising canine variable domains and rodent constant domains from theB lymphocyte cell of claim
 15. 17. A hybridoma cell derived from a Blymphocyte of claim
 15. 18. A part of or whole immunoglobulin moleculecomprising canine variable domains and rodent constant domains from thehybridoma cell of claim 17.